Delivery compositions, and methods of making and using same

ABSTRACT

This invention provides compositions comprising at least one protein nanoparticle comprising a protein and a stealth polymer. In certain embodiments, the nanoparticle further comprises a therapeutic agent, such as a miRNA and/or siRNA. In other embodiments, the nanoparticle further comprises a cell surface receptor ligand. Also included in the invention are methods of preparing the compositions of the present invention, and methods of treating, ameliorating or preventing a disease or disorder in a subject in need thereof using the compositions of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 16/340,864, filed Apr. 10, 2019, nowallowed, which is a 35 U.S.C. § 371 national phase application from, andclaims priority to, International Application No. PCT/US2017/056662,filed Oct. 13, 2017, and published under PCT Article 21(2) in English,which claims priority to U.S. Provisional Application No. 62/407,604,filed Oct. 13, 2016, all of which are incorporated herein by referencein their entireties.

SEQUENCE LISTING

The XML file named “205961-7016U52(00310) Sequence Listing.xml” createdon Oct. 20, 2022, comprising 11 Kbytes, is hereby incorporated byreference in its entirety.

BACKGROUND OF INVENTION

Proteins are chief players in cellular metabolism, being regularly usedas therapeutic agents in medicine. Native or recombinant proteins, alongwith monoclonal antibodies (mAbs), are examples of therapeuticallyrelevant proteins. However, proteins in general are poorly absorbedacross biological membranes, and are thus generally deliveredintravenously. Parenteral administration route has severaldisadvantages, including patient discomfort, potential high cost, andthe risk of needle-stick injuries. Pulmonary administration route offersan excellent alternative for proteins (especially antibodies) targetedtowards local lung diseases. Monoclonal antibodies such as bevacizumab,anatumomab, benralizumab, enokizumab, mitumomab, oxelumab andpalivizumab have gained FDA approval for the treatment of lung diseases,such as asthma, lung cancers and respiratory syncytial virus infection.

Microparticles (rather than nanoparticles) are the particles of choicefor delivering drugs (including proteins) to the lung by inhalation, dueto the widespread belief that nanoparticles are in a size range notsuitable for deep lung delivery. On the other hand, nanoparticles inpulmonary drug delivery may offer advantages such as: (1) potential toachieve relatively uniform distribution of drug dose among the alveoli;(2) enhanced solubility of the drug as compared to its aqueoussolubility; (3) decreased incidence of side effects; (4) improvedpatience compliance; and (5) potential of drug internalization by cells.

Monoclonal antibodies (mAbs) currently do not benefit fully from theunique advantages offered by nanosystems in pulmonary drug delivery,mainly because of the labile nature of their higher order structures,which are not compatible with stresses involved in nanoparticlefabrication.

Bevacizumab, a humanized mAb against vascular endothelial growth factor(VEGF), has shown encouraging signs in the treatment of non-small celllung cancer (NSCLC) when used alone or in combination with chemotherapy.It was approved in 2006 for use along with paclitaxel and carboplatin asfirst-line treatment for those with advanced NSCLC. Cancer cells tend tooverexpress VEGF, a potent stimulator of angiogenesis, facilitatingcancer growth and metastasis. Internalization of bevacizumab into cancercells is highly important, as intracellular pool of VEGF could beresponsible for resistance to bevacizumab in cancer therapy. To thisend, intracellular VEGF provides a compelling target for mAbs in cancertherapy.

RNA interference (RNAi) is a very effective tool in the knockdown ofspecific oncogenes in cancer cells. siRNA is the most widely studiedform of RNAi, and has a promising therapeutic potential in cancer andother diseases such as autoimmune diseases and infectious diseases.Nevertheless, challenges still occur in the development of siRNA as atherapeutic agent due to siRNA's susceptibility to enzymatic degradationin blood, non-specific uptake by cells, and the difficulty involved inits transfection into cells due to its relatively large size andpolarity. Clearance by the reticulo-endothelial system (RES) is anotherlimiting factor affecting the possible therapeutic application of siRNA.

To achieve an efficient knockdown by siRNA, various types of deliverysystems have been investigated. Use of viral vectors are hampered by thepossibility of viral toxicity and immunogenic and inflammatoryreactions. Non-viral vectors such as lipid-based nanoparticles andmesoporous silica are being investigated as possible delivery systemsfor efficient siRNA transfection. In order to achieve an efficientdelivery of siRNA, the delivery system must have the followingproperties: protect siRNA from nuclease degradation duringtransportation in systemic circulation; have minimal RES uptake, therebyallowing for long blood circulation time; allow for effective endosomalescape following internalization by host cells; and most important, mustnot elicit immunological and inflammatory reaction. Lipid nanoparticlesdemonstrate major limitations: siRNA delivery by lipid-basednanoparticles is substantially reduced, because approximately 70% of theinternalized siRNA undergoes exocytosis through egress of the lipidnanoparticles from late endosomes and lysosomes. Use of poly(D,L)-lactide-co-glycolide (PLGA) nanoparticles to deliver siRNA is alsoproblematic, because this polymer is negatively charged and interactsminimally with negatively charged siRNA, thus reducing cellularinternalization. There is thus a growing need for a smart nanoparticledelivery system for efficient and stable siRNA transfection.

MicroRNAs (miRNAs) are frequently utilized as research tools within thebroad borders of gene therapy and the emerging field of molecularmedicine. Although most of the miRNAs are in early stages of clinicaltrials, these classes of compounds have emerged in recent years asextremely promising candidates for drug therapy to a wide range ofdiseases, including cancer, infectious diseases, diabetes,cardiovascular, inflammatory, and neurodegenerative diseases, cysticfibrosis, hemophilia, and other genetic disorders. Due to their shortsequence, miRNA are often able to form a perfect base-pairing withtarget messenger RNA (mRNA) to mediate mRNA degradation. Often, onemiRNA is able to bind to a number of mRNA transcripts enabling theblockade of numerous pathways that modulate cell proliferation,differentiation, apoptosis and invasion.

miRNA-29b is an epi-miRNA that targets DNA methyltranferases (DNMTs) andregulates DNA demethylation, thus leading to down-regulation of globalDNA methylation in malignant cells. Specifically, down-regulation ofDNMT3B led to inhibition of cell proliferation and apoptosis ofnon-small cell lung cancer (NSCLC) cells. In view of this, miRNA-29b isseen as an attractive candidate for miRNA-based therapeutics in NSCLCdue to its potent tumor suppressant capabilities. They have the abilityto silence critical molecular pathways and possibly enhance thesensitivity of NSCLC to conventional chemotherapeutic agents. However,translational application of this relatively novel therapeutic islimited by many challenges, including degradation in serum, rapid bloodclearance, stimulation of immune response, off-target effects, and poorcellular uptake. In order to fully harness this treatment modality inNSCLC, a smart nanoparticle delivery system must be developed in tandemwith the development of miRNAs.

Presently, nanoparticulate systems used in drug delivery include:polymer-based drug carriers (including polymeric nanospheres, polymericmicelles and dendrimers), liposomes, viral nanoparticles, and carbontubes. The processes involved in the fabrication of these nanoparticlesoften lead to degradation and sometimes loss of biological activity inthe biological agent. Further, some materials used to formulate thesenanoparticles may have toxic effects and may not be viable intherapeutic treatments.

There is thus a need in the art for novel and versatile deliverycompositions that are compatible with biological systems and therapeuticagents. There is a further need for novel compositions and formulationsthat allow for pulmonary delivery of therapeutically useful proteins,such as native or recombinant proteins or monoclonal antibodies. Thereis a further need for novel compositions and formulations that allow forefficient and stable miRNA or siRNA transfection. The present inventionsatisfies these needs.

BRIEF SUMMARY OF THE INVENTION

The invention provides a protein-containing nanoparticle, as well as acomposition comprising the same, as well as a method of making the same.The invention further provides a method of treating, ameliorating orpreventing a disease or disorder in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of a nanoparticle and/or a composition of the invention. Theinvention further provides a kit comprising a composition comprising ananoparticle and/or a composition of the invention. The inventionfurther provides a kit comprising a stealth polymer, a proteinconjugated with a cell surface receptor ligand, an applicator, and aninstructional material for the use of the kit.

In certain embodiments, the core of the nanoparticle comprises at leastone protein selected from the group consisting of a plasma protein, anIgG, a cytokine, an immunomodulator, an antigen, a hormone, and anenzyme. In other embodiments, the at least one protein is in a neutralstate in the nanoparticle. In yet other embodiments, the nanoparticle issurrounded by a layer comprising a stealth polymer. In yet otherembodiments, the at least one protein is conjugated with at least onecell surface receptor ligand, wherein at least a fraction of the ligandis displayed on the outer surface of the surrounding layer of thenanoparticle.

In certain embodiments, the stealth polymer is at least one selectedfrom the group consisting of an alkyl polyethylene glycol, analkylphenol oxide, a copolymer of polyethylene glycol and polypropyleneoxide, a polyethylene glycol, a polypropylene glycol, apolyvinylpyrrolidone (PVP), a polyvinyl alcohol, or any combinationsthereof.

In certain embodiments, the nanoparticle has a diameter ranging fromabout 10 nm to about 1,000 nm. In other embodiments, the nanoparticlehas a diameter ranging from about 100 nm to about 500 nm.

In certain embodiments, the plasma protein is at least one selected fromthe group consisting of albumin, fibrinogen, and globulin. In otherembodiments, the cytokine comprises at least one selected from the groupconsisting of interleukin, erythropoietin, interferon, and filgrastim.In yet other embodiments, the protein comprises IgG. In yet otherembodiments, the gG is human.

In certain embodiments, the protein nanoparticle is prepared by a methodcomprising: adjusting the pH of a first solution comprising a protein toabout the isoelectric point of the protein, thereby forming a firstprotein nanoparticle, which comprises at least a fraction of theprotein; wherein, if the protein in the first protein nanoparticle isnot conjugated to at least one cell surface receptor ligand, the proteinin the first protein nanoparticle is further conjugated with the atleast one cell surface receptor; and contacting the first proteinnanoparticle with a second solution comprising a stealth polymer,wherein the concentration of the stealth polymer in the second solutionranges from about 0.1% to about 20,000% of the CMC of the stealthpolymer, thereby forming at least one protein nanoparticle.

In certain embodiments, the first solution further comprises at leastone therapeutic agent, and wherein the first protein nanoparticlecomprises at least a fraction of the at least one therapeutic agent. Inother embodiments, the at least therapeutic agent is selected from thegroup consisting of an organic compound, inorganic compound,pharmacological drug, antibody, radiopharmaceutical, protein, peptide,polysaccharide, nucleic acid, siRNA, miRNA, RNAi, short hairpin RNA,antisense nucleic acid, ribozyme and dominant negative mutant. In yetother embodiments, the at least one therapeutic agent comprises a miRNAor siRNA. In yet other embodiments, the antibody comprises a monoclonalantibody. In yet other embodiments, the monoclonal antibody comprises atleast one selected from the group consisting of bevacizumab, anatumomab,benralizumab, enokizumab, mitumomab, oxelumab, and palivizumab.

In certain embodiments, at least a fraction of the at least one cellsurface receptor ligand is displayed on the outer surface of the stealthpolymer coating of the nanoparticle. In other embodiments, the at leastone cell surface receptor ligand binds to at least one selected from thegroup consisting of neurotensin receptor-1, human epidermal growthfactor receptor-2 (HER-2), folate receptor, insulin-like growth receptor(IGF), and epidermal growth factor receptor (EGFR).

In certain embodiments, the stealth polymer comprises at least oneselected from the group consisting of an alkyl polyethylene glycol, analkylphenol oxide, a copolymer of polyethylene glycol and polypropyleneoxide, a polyethylene glycol, a polypropylene glycol, apolyvinylpyrrolidone (PVP), a polyvinyl alcohol, or any combinationsthereof. In other embodiments, the alkyl polyethylene oxide comprises atleast one selected from the group consisting of a diethylene glycolhexadecyl ether, polyethylene glycol oleyl ether, diethylene glycoloctadecyl ether, polyoxyethylene stearyl ether, polyethylene glycolhexadecyl (cetyl) ether, polyethylene glycol dodecyl (lauryl) ether,decaethylene glycol oleyl ether, polyethylene glycol octadecyl ether,and polyethylene glycol octadecyl ether.

In certain embodiments, the average diameter of the at least one proteinnanoparticle ranges from about 10 nm to about 1,000 nm. In otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 100 nm to about 900 nm. In yet otherembodiments, the concentration of the stealth polymer in the secondsolution ranges from about 100% to about 20,000% of the CMC of thestealth polymer. In yet other embodiments, the concentration of thestealth polymer in the second solution ranges from about 300% to about10,000% of the CMC of the stealth polymer. In yet other embodiments, theconcentration of the stealth polymer in the second solution ranges fromabout 300% to about 5,000% of the CMC of the stealth polymer. In yetother embodiments, the composition further comprises a pharmaceuticallyacceptable carrier.

In certain embodiments, the method of preparing at least one stealthpolymer-coated protein nanoparticle comprises adjusting the pH of afirst solution comprising a protein to about the isoelectric point ofthe protein, thereby forming a first protein nanoparticle, whichcomprises at least a fraction of the protein. In other embodiments, ifthe protein in the first protein nanoparticle is not conjugated to atleast one cell surface receptor ligand, the protein in the first proteinnanoparticle is further conjugated with the at least one cell surfacereceptor. In yet other embodiments, the method of preparing at least onestealth polymer-coated protein nanoparticle comprises contacting thefirst protein nanoparticle with a second solution comprising a stealthpolymer, wherein the concentration of the stealth polymer in the secondsolution ranges from about 0.1% to about 20,000% of the CMC of thestealth polymer.

In certain embodiments, the at least one stealth polymer-coated proteinnanoparticle is further purified to remove protein or stealth polymerthat is not associated with the at least one stealth polymer-coatedprotein nanoparticle, thereby generating a composition comprising the atleast one stealth polymer-coated protein nanoparticle. In otherembodiments, the composition comprising at least stealth polymer-coatednanoparticle is further lyophilized.

In certain embodiments, the method of treating, ameliorating orpreventing a disease or disorder in a subject in need thereof comprisesadministering to the subject a pharmaceutically effective amount of ananoparticle or composition of the invention, further wherein thecomposition is administered to the subject by an intrapulmonary,intrabronchial, inhalational, intranasal, intratracheal, intravenous,intramuscular, subcutaneous, topical, transdermal, oral, buccal, rectal,pleural, peritoneal, vaginal, epidural, otic, intraocular, orintrathecal route. In other embodiments, the composition is administeredto the subject by an intrapulmonary, intrabronchial, inhalational,intranasal, intratracheal, intravenous, intramuscular, subcutaneous ortopical route. In yet other embodiments, the composition furthercomprises at least one therapeutic agent, which is within the proteinnanoparticle. In yet other embodiments, the at least therapeutic agentis selected from the group consisting of an organic compound, inorganiccompound, pharmacological drug, antibody, radiopharmaceutical, protein,peptide, polysaccharide, nucleic acid, siRNA, RNAi, short hairpin RNA,antisense nucleic acid, ribozyme and dominant negative mutant. In yetother embodiments, the at least therapeutic agent comprises a siRNA ormiRNA.

In certain embodiments, the protein comprises IgG. In other embodiments,the nanoparticle further comprises a therapeutic agent comprising asiRNA or a miRNA. In yet other embodiments, the stealth polymercomprises a copolymer of polyethylene oxide and polypropylene oxide. Inyet other embodiments, the at least one cell surface receptor ligandbinds to at least one selected from the group consisting of neurotensinreceptor-1, human epidermal growth factor receptor-2 (HER-2), folatereceptor, insulin-like growth receptor (IGF), and epidermal growthfactor receptor (EGFR). In yet other embodiments, the disease ordisorder is selected from the group consisting of colon cancer, rectumcancer, lung cancer, glioblastoma, renal cell cancer, non-small celllung cancer, small cell lung cancer, asthma, respiratory syncytial virus(RSV) infection, and any combinations thereof. In yet other embodiments,the disease or disorder comprises a cancer comprising a KRAS mutation.In yet other embodiments, the subject is a mammal. In yet otherembodiments, the mammal is human.

In certain embodiments, the kit further comprises an applicator; and aninstructional material for the use of the kit, wherein the instructionmaterial comprises instructions for treating, ameliorating or preventinga disease or disorder in a subject in need thereof. In otherembodiments, the stealth polymer-containing protein nanoparticle furthercomprises at least one therapeutic agent.

In certain embodiments, the instruction material comprises instructionsfor preparing a stealth polymer-coated protein nanoparticle wherein atleast a fraction of the ligand is displayed on the outer surface of thestealth polymer coating of the nanoparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the present invention. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIGS. 1A-1E comprises a set of images and graphs illustrating hybridnanoparticle characterization. FIG. 1A comprises a non-limitingschematic of an aptamer-functionalized hybrid nanoparticle. FIG. 1Bcomprises a scanning electron micrograph of miRNA-loaded hybridnanoparticles (scale bar: 500 nm). FIG. 1C comprises a fluorescencemicrograph of FITC-MUC1 aptamer-functionalized hybrid nanoparticles(scale bar: 25 μm). FIG. 1D comprises a fluorescent micrograph ofnon-functionalized hybrid nanoparticles (scale bar: 25 μm). FIG. 1Ecomprises FT-IR spectra of various nanoparticles to confirm theconjugation of aptamer to the nanoparticles.

FIG. 2 comprises a graph illustrating in vitro release. Release profilesshow that only a limited amount of microRNA-29b was released at pHvalues 6.6 and 7.4. However, an optimal release was observed at pH 5.

FIGS. 3A-3C comprises a series of images and graphs illustrating impactof MUC1 expression levels on MUC1 aptamer-functionalized hybridnanoparticle internalization. FIG. 3A illustrates reverse transcriptasePCR showing levels of expression of MUC1 in selected cells (n=3). FIG.3B illustrates comparison of nanoparticle internalization by A549 andMRC-5 using flow cytometry. **P≤0.001, n=3. FIG. 3C is a graphillustrating mean fluorescence intensity for non-inhibited nanoparticlesand MUC1-inhibited nanoparticles.

FIGS. 4A-4B comprise a series of images illustrating fluorescencemicroscopy of nanoparticle-cell membrane interaction. FIG. 4A comprisesa series of micrographs showing the interaction between FITC-MUC1aptamer-functionalized nanoparticles and cell membrane after 2-hourincubation. FIG. 4B comprises a series of micrographs showingintracellular trafficking of internalized siGLO-FAM-loadednanoparticles.

FIGS. 5A-5D comprises a series of images and graphs illustrating effectof miRNA-29b on essential oncoproteins in A549 cells. FIG. 5Aillustrates downregulation of DNMT3B by miRNA-29b nanoparticles. FIG. 5Billustrates downregulation of MCL1 by miRNA-29b nanoparticles.miR-29b-nano represents MUC1 aptamer-functionalized miRNA-29b-loadedhybrid nanoparticles; NC-nano represents MUC1 aptamer-functionalizednegative control miRNA-loaded hybrid nanoparticles; miR-29b-liporepresents lipofectamine 200-transfected miRNA-29b. FIG. 5C illustratescell death detection ELISA showing the induction of apoptosis in A549cells (n=3). FIG. 5D illustrates antiproliferative effect of miRNA-29bnanoparticles in A549 cells (n=5).

FIG. 6 illustrates preparation and characterization of mucin1-aptamerfunctionalized miRNA-29b-loaded hybrid nanoparticles (MAFMILHN).Schematic showing the procedure involved in the production of MAFMILHN.

FIGS. 7A-7H illustrate hyperspectral imaging of nanoparticleinternalization. FIG. 7A: MUC1-aptamer functionalized miR-29b-loadedhybrid nanoparticles (MAFMILHN). FIG. 7B: non-functionalizedmiR-29b-loaded hybrid nanoparticles. FIG. 7C: untreated A549 cells. FIG.7D: untreated MRC-5 cells. FIG. 7E: MAFMILHN-treated A549 cells. FIG.7F: non-functionalized miR-29b-loaded hybrid nanoparticle-treated A549cells. FIG. 7G: MAFMILHN-treated MRC-5 cells. FIG. 7H:non-functionalized miR-29b-loaded hybrid nanoparticle-treated MRC-5cells. Arrows depict MAFMILHN in cells.

FIGS. 8A-8D illustrate tissue distribution of MAFMILHN in the lung oftumor bearing mice. FIG. 8A: MAFMILHN. FIG. 8B: Non-functionalizedmiR-29b-loaded hybrid nanoparticles. FIG. 8C: Hyperspectral imaging ofMAFMILH. FIG. 8D: Hyperspectral imaging of Non-functionalizedmiR-29b-loaded hybrid nanoparticles. Scale bar=10 μm.

FIG. 9 is a graph illustrating miRNA-27b plasma concentration over time.Tumor bearing mice were given a single dose of 1.5 mg/kg of miRNA-29b inMAFMILHN or in non-functionalized miRNA-29b-loaded hybrid nanoparticles.n=3.

FIGS. 10A-10B illustrate apoptosis in treated tumor-bearing lungs. FIG.10A: Evaluation of apoptosis using TUNEL. FIG. 10B: Evaluation ofapoptosis using cell death detection ELISA, ***p≤0.001, n=3.

FIGS. 11A-11B illustrate in vivo antitumor effect in SCID beige micemonitored using IVIS bioluminescence imaging system. FIG. 11A: Graph oftumor burden over four week period. FIG. 11B: Representativebioluminescence images of tumor-bearing mice.

DETAILED DESCRIPTION OF INVENTION

The present invention relates in part to the unexpected discovery ofnovel protein nanoparticles, as well as methods of making the same. Inone aspect, the methods of the present invention allow for carefulcontrol of the content, size and shape of the nanoparticles, thusenhancing their overall drug delivery properties.

In certain embodiments, the methods of the present invention providenanoparticles with distinct compositions, improved loading capacity,and/or lower particle size as compared to the methods disclosed inUS20160213777, which is incorporated herein in its entirety byreference.

In certain embodiments, particle sizes of the nanoparticles obtainedusing the methods of the present invention are smaller than thoseobtained with the methods of the prior art. In other embodiments,particle sizes of the nanoparticles obtained using the methods of thepresent invention are equal to or lower than 1,000 nm, 900 nm, 800 nm,700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 190 nm, 180 nm, 170 nm,160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70nm, 60 nm, and/or 50 nm.

In certain embodiments, the nanoparticle of the invention comprises acore comprising a protein (such as but not limited to a plasma protein,an IgG, a cytokine, an immunomodulator, an antigen, a hormone, and/or anenzyme), which is in a neutral state (i.e., the overall charge of theprotein within the nanoparticle is zero or nearly zero). In otherembodiments, the nanoparticle of the invention is surrounded by astealth polymer layer (or stealth polymer coating), which comprises forexample an alkyl polyethylene glycol, an alkylphenol oxide, a copolymerof polyethylene glycol and polypropylene oxide (such as, but not limitedto, a poloxamer), a polyethylene glycol, a polypropylene glycol, apolyvinylpyrrolidone (PVP), a polyvinyl alcohol, or any combinationsthereof. In yet other embodiments, the nanoparticle of the inventioncomprises at least one cell surface receptor ligand, wherein at least afraction of the ligand is displayed on the surface of the nanoparticle.In yet other embodiments, at least a portion of the cell surfacereceptor ligand spans the stealth polymer layer. In yet otherembodiments, the at least one cell surface receptor ligand is conjugatedto the protein via non-covalent bond(s). In yet other embodiments, theat least one cell surface receptor ligand is conjugated to the proteinvia covalent bond(s). In yet other embodiments, the at least one cellsurface receptor ligand is conjugated to the protein before the proteinis coated with the stealth polymer. In yet other embodiments, the atleast one cell surface receptor ligand is conjugated to the proteinbefore the protein nanoparticle is formed. In yet other embodiments, theat least one cell surface receptor ligand is conjugated to the proteinafter the protein nanoparticle is formed, but before the protein iscoated with the stealth polymer.

In certain embodiments, the nanoparticles of the invention comprise apoloxamer, which helps achieve stable and efficient delivery of nucleicacid-based therapeutics. In other embodiments, the core of thenanoparticles comprises a protein, such as but not limited to human IgG,which is a main encapsulating component of these hybrid nanoparticles.Without wishing to be limited by any theory, the protein helps toreduce/eliminate well-documented immunogenic reaction experienced withmost nanoparticle formulations, by “deceiving” the body to believe thatthe nanoparticles are natural components of the blood. In yet otherembodiments, the outer layer of these hybrid nanoparticles is composedof poloxamer, a nonionic triblock copolymer, which helps to circumventthe reticuloendothelial system during systemic circulation, acting as astealth polymer by preventing macrophage uptake during circulation.

In certain embodiments, the nanoparticles of the invention arefunctionalized on their surface with at least one cell surface receptorligand, such as, but not limited to a MUC1-aptamer. In otherembodiments, the ligand facilitates active targeting of the nanoparticleto the surface receptor-expressing cells, while avoiding undesirableaccumulation in cells that do not express such surface receptors.

In certain embodiments, a method of the invention comprises providing asolution comprising a protein of interest, and optionally at least onetherapeutic agent, and titrating the solution to about the isoelectricpoint of the protein, thereby forming a precipitate comprising theprotein nanoparticle, wherein the protein nanoparticle comprises atleast a fraction of the protein (and at least a fraction of thetherapeutic agent, if the at least one therapeutic agent is present inthe solution). The protein nanoparticle is optionally purified; in anon-limiting example, the protein nanoparticle is washed with a solvent,such as but not limited to water. The protein nanoparticle is thenresuspended in a solution comprising a stealth polymer, wherein theconcentration of the stealth polymer in the solution ranges from about0.1% to about 20,000% of the CMC of the stealth polymer, and optionallyat least one therapeutic agent. The resulting stealth polymer-containingprotein nanoparticle can then be purified and/or isolated from thesolution. Optionally, a cell surface receptor ligand is present in theprotein nanoparticle and/or the stealth polymer-coated proteinnanoparticle.

In certain embodiments, the nanoparticles of the present invention haveimproved aerosolization properties as compared to irregularly shaped(>20 μm) unprocessed particles. In other embodiments, the nanoparticlesof the present invention comprise at least one therapeutic agent,wherein the at least one therapeutic agent within the nanoparticles hasimproved pharmacokinetics as compared to the “unformulated” therapeuticagent (i.e., the therapeutic agent that is not within the nanoparticlesof the present invention). In yet other embodiments, the nanoparticlesof the present invention further comprise a cell surface receptorligand, which allows for the nanoparticles to recognize and bind to acell that displays such cell surface receptor.

In certain embodiments, the protein comprises a native or recombinanttherapeutically useful protein, such as plasma proteins like albumins,fibrinogen and clotting factors; hormones like insulin, glucagon, andsomatropin; immunomodulators like cyclosporine; cytokines likeinterleukin, erythropoietin, interferon, and filgrastim; enzymes likeblood clotting factors, adenosine deaminase, alpha1 antitryptin; andpeptide vaccines. In other embodiments, the protein comprises anantibody. In yet other embodiments, the protein comprises animmunoglobulin. In yet other embodiments, the immunoglobulin comprisesIgA, IgD, IgE, IgG, and/or IgM. In yet other embodiments, the antibodycomprises a monoclonal antibody (mAb).

Potential applications of the mAb nanoparticles of the present inventioninclude, in a non-limiting manner, selective targeting of intracellularoncoproteins in cancer; pulmonary delivery of mAb by dry powderinhalation and pressurized metered dose inhalation and nebulizers; as acarrier system for delivering nucleic acids and/or small molecule tocells; and formulation of high concentration mAb dosage forms forvarious diseases.

Non-Limiting Disclosure

The therapeutic efficacy and pharmacokinetics of mucin1-aptamerfunctionalized miRNA-29b-loaded hybrid nanoparticles (MAFMILHN) in lungtumor-bearing SCID mice was evaluated, as described herein. MAFMILHNwere manufactured using an iso-electric point based nanotechnology. Theywere then fully characterized for particle size, Zeta potential, loadingcapacity and encapsulation efficiency. The ability of MAFMILHN todownregulate oncoprotein DNMT3B both at the cellular level and in vivowas monitored using western blot, while the effect of the downregulationof DNMT3B was assessed using bioluminescence. Results indicate that thepresence of MUC1-aptamer on the surface of the nanoparticles enhancedthe selective delivery of miRNA-29b to tumor cells and tissues. Further,the downregulation of DNMT3B by MAFMILHN resulted to the inhibition oftumor growth in mouse models. The present studies indicate that theMAFMILHNs selectively deliver miRNA-29b to lung tumor while limitingaccumulation in healthy tissues. In certain non-limiting embodiments,very limited expression of MUC1 in healthy tissues enables selectiveaccumulation of miRNA-29b loaded hybrid nanoparticles in NSCLC whileavoiding healthy tissues. In other non-limiting embodiments, delivery ofMAFMILHNs lead to in vivo downregulation of DNMT3B, leading toregression of NSCLC in orthotopic mouse models.

Translational application of miRNA-based therapeutics is limited by alack of smart nanoparticle delivery system to selectively deliver thesemolecules intracellularly to cancer cells. As a demonstrated herein, ahybrid nanoparticles delivery system, which is capable of safe andeffective delivery of nucleic acid-based therapeutics, wasfunctionalized with MUC1 aptamer to enable selective delivery of miRNAsto NSCLC cells. miRNA-29b was selected as a model miRNA, becausemiRNA-29b, a tumor suppressor miRNA, is aberrantly expressed in NSCLCand its perturbation is related to tumor development and progression.miRNA-29b was thus an attractive candidate for miRNA-based therapeuticsin NSCLC. The presence of MUC1, a transmembrane protein that isaberrantly overexpressed in NSCLCs, allows for the active targeting ofpayload to lung adenocarcinomas. MUC1 has been shown to be overexpressedin 80% of lung adenocarcinoma.

Hybrid nanoparticles comprising human IgG and poloxamer-188 wereprepared using an isoelectric point (pI)-based nanoprecipitationtechnology. This technology is based on the fact that proteins (humanIgG) have minimum solubility but maximum precipitation at their pI.Nanoparticles produced using this technology are characteristicallyspherical in morphology (FIG. 1B). The particles were negatively chargedpossibly because of the presence of residual miRNA on the surface of thenanoparticles. However, the charge on the particles became positivefollowing the conjugation of aptamer to the surface of thenanoparticles. Without wishing to be limited by any theory, the positivecharge is attributed at least in part to the presence of an amino groupNH₂ in the aptamer to facilitate the conjugation reaction. Bothfluorescence microscopy and FT-IR analysis were performed to confirm thesuccessful conjugation of MUC1 aptamer to the preformed hybridnanoparticles. As shown in FIG. 1C, FITC-labeled MUC1 aptamer present onthe surface of the hybrid nanoparticles led to the presence of greencolor in the MUC1 aptamer-functionalized hybrid nanoparticles. Incontrast, non-functionalized hybrid nanoparticles did not show any greencolor in FIG. 1D. FT-IR data in FIG. 1E demonstrate a distinctivedifference between the spectra of MUC1 aptamer-functionalized hybridnanoparticles and the non-functionalized hybrid nanoparticles.Non-functionalized hybrid nanoparticles had a peak at about 3,274 cm⁻¹,which can be attributed to the group stretching vibration of both NH₂and OH in the human IgG. Following the conjugation of MUC1 aptamer tothe hybrid nanoparticles, this peak became sharper and more intense dueto the presence of additional NH₂ and OH stretching vibrationattributable to the conjugation of NH₂ aptamer to the hybridnanoparticles. Both MUC1 aptamer-functionalized and non-functionalizednanoparticles showed a peak at about 1,541 cm⁻¹, which can be attributedto the presence of amide II carbonyl stretch from human IgG in bothnanoparticles. Furthermore, without wishing to be limited by any theory,the peak at about 1,660 cm⁻¹ in the spectra for MUC1aptamer-functionalized hybrid nanoparticles, but absent in thecorresponding non-functionalized hybrid nanoparticles, is attributed toconjugated amide stretching due to the conjugation of the amino groupfrom the aptamer to the carboxyl groups present in human IgG.

FIG. 2 demonstrates a limitation in the release of miRNA from MUC1aptamer-functionalized hybrid nanoparticles at pH values 6.6 and 7.4when compared with the release profile at pH 5. This demonstrates thepH-sensitive nature of these nanoparticle delivery systems. At pH valuesof 6.6 and 7.4, very limited amount of the loaded miRNA (<20%) wasreleased throughout the study period. The limited release of siRNA at pHvalues 6.6 and 7.4 could be attributed to the reduced/limited solubilityof human IgG at these pH values. Proteins are known to have limitedsolubility at pH values close to their pI. Since the pI of human IgG is7, its solubility at neutral pH values is quite limited. This makes itdifficult for the encapsulated miRNA to be released at this pH value,hence possibly limiting its release extracellularly in blood and tumormicroenvironment. However, at acidic pH value of 5, an optimal miRNArelease of about 100% was obtained due to the solubility of human IgG atthis pH. An optimum release of the loaded miRNA is very desirable at pH5, as this pH represents the acidic condition of endosome/lysosome.

Uptake of MUC1 aptamer-functionalized nanoparticles (FIG. 3B) issignificantly higher in A549 cells in comparison to MRC-5 cells. Withoutwishing to be limited by any theory, the differential internalization ofnanoparticles between these cell lines can be attributed at least inpart to the differential expression of MUC1 on the surface of thesecells. While A549 is an adenocarcinoma cell line known to aberrantlyoverexpress MUC1, MRC-5 is a normal lung fibroblast cell line withlimited expression of MUC1 (FIG. 3A). To further confirm the involvementof MUC1 in the internalization of these nanoparticles, cells werepretreated with free MUC1 aptamer prior to their treatment with the MUC1aptamer-functionalized hybrid nanoparticles to further elucidate therole of MUC1 in uptake of nanoparticles. In FIG. 3B, the significantlylower uptake of nanoparticles by A549 cells following pre-treatment withfree MUC1 aptamer can be attributed at least in part to competitivebinding of this free MUC1 aptamer to MUC1 on the membrane of the cells,limiting the uptake of MUC1 aptamer-functionalized hybrid nanoparticles.FIG. 4A further demonstrates the interaction between the cell membraneof A549 cells and FITC-MUC1 aptamer conjugated to the hybridnanoparticles. Presence of green color on the membrane of the A549 cellssuggests the binding of these nanoparticles to the membrane prior tointernalization.

One of the challenges facing the translational application ofnanomedicine is the lack of effective endosomal escape followinginternalization by host cells. Stable nucleic acid lipid particles,which to date are the most advanced delivery system for nucleic acid,have demonstrated some limitation in recent studies. About 70% of theinternalized nucleic acid undergoes exocytosis (endocytic recycling)through egress of the lipid nanoparticles from late endosomes andlysosomes. It is thus important to design nanoparticle delivery systemwith the ability to escape the recycling pathways. To demonstrate theability of the hybrid nanoparticles to escape endocytic recycling,intracellular trafficking of internalized nanoparticles was monitoredusing fluorescence microscopy. In FIG. 4B, colocalization ofnanoparticles (green) and endosome (red) indicates the presence ofnanoparticles in the late endosome after 2 hours of incubation. However,release of siGLO-FAM (green) can be seen after 4 hours of incubation,indicating the escape of encapsulated siGLO-FAM from late endosomes intothe cytosol of the cells. This was possible due to the bufferingcapacity of the hybrid nanoparticles in the endosome. The solubility ofIgG and poloxamer-188 at acidic pH 5, as demonstrated by the in vitrorelease data in FIG. 2 , makes it possible for the dissolved IgG in theendosome to activate the proton pump that raises osmotic pressure in theendosome subsequently leading to the swelling and subsequent escape ofsiGLO-FAM from the endosomes into the cytoplasm.

To evaluate the ability of internalized miRNA-29b to downregulate targetoncoproteins DNMT3B and MCL1, expression of these proteins was monitoredusing Western blot. As demonstrated in FIGS. 5A-5B, both targetoncoproteins were downregulated in A549 cells following treatment withMUC1 aptamer-functionalized miRNA-29b-loaded hybrid nanoparticles in asuperior version when compared with lipofectamine-transfected miRNA-29b.DNMT3B is a member of the DNA methyltransferase family accounting forinactivation of tumor suppressor genes in many cancer cells. miRNA-29bexerts its tumor-suppressive role by directly targeting DNMT3b in cancercells. Furthermore, miRNA-29b is downregulated in malignant cells,whereas MCL1 is upregulated. MCL1 is a potent antiapoptic protein of theBCL-2 family. Downregulation of both DNMT3B and MCL1 by miRNA-29binhibits cancer cell growth and promotes apoptosis. However, thechallenge facing the clinical application of miRNA-29b to cancer therapyis the availability of an efficient and safe delivery system to enhanceintracellular delivery of this potent molecule to cancer cells. FIGS.5A-5B demonstrates the superior efficiency of MUC1aptamer-functionalized miRNA-29b-loaded hybrid nanoparticles indownregulating oncoproteins DNMT3b and MCL1 in NSCLC cells overlipofectamine-delivered miRNA-29b. The ability of this treatmentmodality to induce apoptosis in A549 cells due to downregulation ofDNMT3B and MCL1 was also demonstrated in FIG. 5C. MUC1 aptamermiRNA-29b-loaded hybrid nanoparticles also demonstrated superiorantiproliferative effect in A549 cells in comparison to NeoFX-deliveredmiRNA-29b. NeoFX is another commonly used, commercially availablenucleic acid transfection agent.

As demonstrated herein, MUC1 aptamer-functionalized hybrid nanoparticledelivery system was successfully prepared. The present resultsdemonstrated that this delivery system can efficiently deliver miRNAseffectively to cancer cells in a superior version compared withcommercially available transfection agents. The present results alsodemonstrated that direct downregulation of DNMT3b and MCL1 led toantiproliferative effect in A549 cells. In certain embodiments, thisnovel aptamer-hybrid nanoparticle bioconjugate delivery system can serveas a platform for intracellular delivery of miRNAs to cancer cells,hence improving the therapeutic outcome of lung cancer.

Definitions

The definitions used in this application are for illustrative purposesand do not limit the scope used in the practice of the presentinvention.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, polymer chemistry, andprotein chemistry are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” are used herein to refer toone or to more than one (i.e., to at least one) of the grammaticalobject of the article. By way of example, “an element” means one elementor more than one element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein, “about” when referring to a measurablevalue such as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The term “antibody” as used herein refers to an immunoglobulin moleculeable to specifically bind to a specific epitope on an antigen.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources, and can be immunoreactive portions of intactimmunoglobulins. The antibodies useful in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)₂, as well as single chain antibodies(scFv), camelid antibodies and humanized antibodies (Harlow et al.,1998, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antigen” or “Ag” is defined as a molecule that provokes animmune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, such as virtually all proteins or peptides, can serve asan antigen. Furthermore, antigens can be derived from recombinant orgenomic DNA. A skilled artisan will understand that any DNA, whichcomprises a nucleotide sequences or a partial nucleotide sequenceencoding a protein that elicits an immune response therefore encodes an“antigen” as that term is used herein. Furthermore, one skilled in theart will understand that an antigen need not be encoded solely by a fulllength nucleotide sequence of a gene. The present invention includes,but is not limited to, the use of partial nucleotide sequences of morethan one gene, and these nucleotide sequences are arranged in variouscombinations to elicit the desired immune response. Moreover, a skilledartisan will understand that an antigen need not be encoded by a “gene”at all. An antigen can be generated, synthesized or derived from abiological sample. Such biological sample can include, but is notlimited to, a tissue sample, tumor sample, cell or biological fluid.

As the term is used herein, “applicator” is used to identify any deviceincluding, but not limited to, a hypodermic syringe, pipette, nebulizer,vaporizer and the like, for administering the compounds and compositionsused in the practice of the present invention.

As used herein, “aptamer” refers to a small molecule that can bindspecifically to another molecule. Aptamers are typically eitherpolynucleotide- or peptide-based molecules. A polynucleotidal aptamer isa DNA or RNA molecule, usually comprising several strands of nucleicacids, that adopts highly specific three-dimensional conformationdesigned to have appropriate binding affinities and specificitiestowards specific target molecules, such as peptides, proteins, drugs,vitamins, among other organic and inorganic molecules. Suchpolynucleotidal aptamers can be selected from a vast population ofrandom sequences through the use of systematic evolution of ligands byexponential enrichment. A peptide aptamer is typically a loop of about10 to about 20 amino acids attached to a protein scaffold that bind tospecific ligands. Peptide aptamers may be identified and isolated fromcombinatorial libraries, using methods such as the yeast two-hybridsystem.

As used herein, the term “BRIJ®” is a trademark that described anon-ionic detergent comprising an oligo- or poly-ethylene glycolmono-derivatized with an aliphatic chain (an alkyl polyethylene oxide).Examples of BRIJ® compounds comprises BRIJ® 52 (polyethylene glycolhexadecyl ether; M_(n) ˜330), BRIJ® 58 (polyethylene glycol hexadecylether; M_(n) ˜1,124), BRIJ® 93 (polyethylene glycol oleyl ether; M_(n)˜357), BRIJ® C10 (polyethylene glycol hexadecyl ether), BRIJ® L4(tetraethylene glycol dodecyl ether), BRIJ® L23 (tricosethylene glycoldodecyl ether), BRIJ® O10 and BRIJ® O20 (decaethylene glycol oleylether), BRIJ® S2 (diethylene glycol octadecyl ether), BRIJ® S10 andBRIJ® S100 (decaethylene glycol octadecyl ether),

As used herein, “biologically active” means that the compositions elicita biological response in a mammal that can be monitored andcharacterized in comparison with an untreated mammal. In certainembodiments, the compositions are administered to the respiratory tractof the mammal. In other embodiments, the compositions are useful forinhalational, nasal, intrapulmonary, intrabronchial, or inhalationadministration. In yet other embodiments, the compositions are usefulfor nasal, inhalational, topical, oral, buccal, rectal, pleural,peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural,intratracheal, otic, intraocular, intrathecal or intravenousadministration.

As used herein, the term “CMC” refers to critical micelle concentration.The CMC of a stealth polymer is defined as the solution concentration ofthe stealth polymer above which stealth polymer micelles formspontaneously. In certain embodiments, additional stealth polymer addedto the system beyond the CMC value is incorporated in more micelles.

As used herein, the term “container” includes any receptacle for holdingthe pharmaceutical composition. For example, in certain embodiments, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well-known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions can contain information pertaining to the compound'sability to perform its intended function.

As used herein, a “disease” is a state of health of an animal whereinthe animal cannot maintain homeostasis, and wherein if the disease isnot ameliorated then the animal's health continues to deteriorate.

As used herein, a “disorder” in an animal is a state of health in whichthe animal is able to maintain homeostasis, but in which the animal'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” of acomposition are used interchangeably to refer to the amount of thecomposition that is sufficient to provide a beneficial effect to thesubject to which the composition is administered.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly five amino acids and/or sugars in size. One skilledin the art understands that generally the overall three-dimensionalstructure, rather than the specific linear sequence of the molecule, isthe main criterion of antigenic specificity and therefore distinguishesone epitope from another.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with an antigen and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of V_(H) (variable heavy chain immunoglobulin) genes from ananimal.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of a composition of thepresent invention in a kit. The instructional material of the kit may,for example, be affixed to a container that contains a composition ofthe present invention or be shipped together with a container whichcontains a composition. Alternatively, the instructional material may beshipped separately from the container with the intention that therecipient uses the instructional material and a compositioncooperatively. Delivery of the instructional material may be, forexample, by physical delivery of the publication or other medium ofexpression communicating the usefulness of the kit, or may alternativelybe achieved by electronic transmission, for example by means of acomputer, such as by electronic mail, or download from a website.

As used herein, the term “IP-particle” or “IP-nanoparticle” refers toIgG-poloxamer-188 nanoparticle.

As used herein, the term “medical intervention” means a set of one ormore medical procedures or treatments that are required for amelioratingthe effects of, delaying, halting or reversing a disease or disorder ofa subject. A medical intervention may involve surgical procedures ornot, depending on the disease or disorder in question. A medicalintervention may be wholly or partially performed by a medicalspecialist, or may be wholly or partially performed by the subjecthimself or herself, if capable, under the supervision of a medicalspecialist or according to literature or protocols provided by themedical specialist.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man is a naturally-occurring sequence.

As used herein, the terms “peptide” and “polypeptide” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise the sequence of aprotein or peptide. Polypeptides include any peptide or proteincomprising two or more amino acids joined to each other by peptidebonds. As used herein, the term refers to both short chains, which alsocommonly are referred to in the art as peptides, oligopeptides andoligomers, for example, and to longer chains, which generally arereferred to in the art as proteins, of which there are many types.“Polypeptides” include, for example, biologically active fragments,substantially homologous polypeptides, oligopeptides, homodimers,heterodimers, variants of polypeptides, modified polypeptides,derivatives, analogs and fusion proteins, among others. The polypeptidesinclude natural peptides, recombinant peptides, synthetic peptides or acombination thereof. A peptide that is not cyclic has a N-terminus and aC-terminus. The N-terminus has an amino group, which can be free (i.e.,as a NH₂ group) or appropriately protected (for example, with a BOC or aFmoc group). The C-terminus has a carboxylic group, which can be free(i.e., as a COOH group) or appropriately protected (for example, as abenzyl or a methyl ester). A cyclic peptide does not necessarily havefree N- or C-termini, since they are covalently bonded through an amidebond to form the cyclic structure.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, a “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation, and notinjurious to the patient. Some examples of materials that can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. As used herein“pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound, and are physiologically acceptable to the subject.Supplementary active compounds can also be incorporated into thecompositions.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof.

As used herein, the term “poloxamer” refers to a non-ionic triblockcopolymer composed of a central hydrophobic chain of polyoxypropylene(also known as poly(propylene oxide)) flanked by two hydrophilic chainsof polyoxyethylene (also known as poly(ethylene oxide)). Poloxamers arealso known by the trade names SYNPERONIC®, PLURONIC®, and KOLLIPHOR®.For the generic term “poloxamer”, these copolymers are commonly namedwith the letter “P” (for poloxamer) followed by three digits, whereinthe first two digits×100 represents the approximate molecular mass ofthe polyoxypropylene core, and the last digit×10 represents thepercentage polyoxyethylene content (e.g., P407 is a poloxamer with apolyoxypropylene molecular mass of 4,000 g/mol and a 70% polyoxyethylenecontent). For the PLURONIC® and SYNPERONIC® trade names, coding of thesecopolymers starts with a letter to define its physical form at roomtemperature [L=liquid, P=paste, F=flake (solid)] followed by two orthree digits. The first digit in a two-digit number, or the first twodigits in a three-digit number, in the numerical designation, multipliedby 300, represents the approximate molecular weight of the hydrophobe;and the last digit×10 represents the percentage polyoxyethylene content(e.g., L61 indicates a polyoxypropylene molecular mass of 1,800 g/moland a 10% polyoxyethylene content). In certain embodiments, poloxamer181 (P181), PLURONIC® L61 and SYNPERONIC® PE/L 61 are interchangeable.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.Disease and disorder are used interchangeably herein.

As used herein, a “prophylactic” or “preventive” treatment is atreatment administered to a subject who does not exhibit signs of adisease or disorder or exhibits only early signs of the disease ordisorder for the purpose of decreasing the risk of developing pathologyassociated with the disease or disorder.

As used herein, the term “RES” refers to reticulo-endothelial system.

By the term “specifically bind” or “specifically binds” as used hereinis meant that a first molecule (e.g., an antibody) preferentially bindsto a second molecule (e.g., a particular antigenic epitope), but doesnot necessarily bind only to that second molecule.

As used herein, the term “stealth polymer” refers to a polymer or asurfactant that can be used to coat a particulate object and reducesopsonization of the coated particulate object by phagocytes of thereticulo-endothelial system. In certain embodiments, the stealth polymercoating reduces in vivo engulfment or clearance of the coatedparticulate object. Non-limiting examples of stealth polymers include analkyl polyethylene glycol, an alkylphenol oxide, a copolymer ofpolyethylene glycol and polypropylene oxide (such as, but not limitedto, a poloxamer), a polyethylene glycol, a polypropylene glycol, apolyvinylpyrrolidone (PVP), a polyvinyl alcohol, or any combinationsthereof.

A “subject” or “individual” or “patient,” as used therein, can be ahuman or non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the subject is human.

By the term “synthetic antibody” as used herein is meant an antibodygenerated using recombinant DNA technology, such as, for example, anantibody expressed by a bacteriophage as described herein. The termshould also be construed to mean an antibody generated by the synthesisof a DNA molecule encoding the antibody and which DNA molecule expressesan antibody protein, or an amino acid sequence specifying the antibody,wherein the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

As used herein, a “therapeutic” treatment is a treatment administered toa subject who exhibits signs of pathology of a disease or disorder forthe purpose of diminishing or eliminating those signs.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringa composition to reduce the severity with which symptoms areexperienced. An appropriate therapeutic amount in any individual casemay be determined by one of ordinary skill in the art using routineexperimentation.

The following abbreviations are used herein: DAPI,4,6-diamidino-2-phenylindole; FT-IR, Fourier transform-infrared; GAPDH,glyceraldehyde 3-phosphate dehydrogenase; IgG, immunoglobulin G; mAb,monoclonal antibody; MAFMILHN, mucin1-aptamer functionalizedmiRNA-29b-loaded hybrid nanoparticle(s); miRNA, microRNA; MUC1, mucin 1.

Throughout this disclosure, various aspects of the present invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thepresent invention. Accordingly, the description of a range should beconsidered to have specifically disclosed all the possible sub-ranges aswell as individual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

Compositions

In certain embodiments, the invention provides a protein-containingnanoparticle, wherein the core of the nanoparticle comprises at leastone protein selected from the group consisting of a plasma protein, anIgG, a cytokine, an immunomodulator, an antigen, a hormone, and anenzyme, wherein the at least one protein is in a neutral state in thenanoparticle, wherein the nanoparticle is surrounded by a layercomprising a stealth polymer, wherein the at least one protein isconjugated with at least one cell surface receptor ligand, wherein atleast a fraction of the ligand is displayed on the outer surface of thesurrounding layer of the nanoparticle.

In certain embodiments, compositions of the invention are prepared usinga method that comprises providing a solution comprising a protein ofinterest, and optionally at least one therapeutic agent, and titratingthe solution to about the isoelectric point of the protein, therebyforming a precipitate comprising the protein nanoparticle, wherein theprotein nanoparticle comprises at least a fraction of the protein (andat least a fraction of the therapeutic agent, if the at least onetherapeutic agent is present in the solution). The protein nanoparticleis optionally purified; in a non-limiting example, the proteinnanoparticle is washed with a solvent, such as but not limited to water.The protein nanoparticle is then resuspended in a solution comprising astealth polymer, wherein the concentration of the stealth polymer in thesolution ranges from about 0.1% to about 20,000% of the CMC of thestealth polymer, and optionally at least one therapeutic agent. Theresulting stealth polymer-containing protein nanoparticle can then bepurified and/or isolated from the solution. Optionally, a cell surfacereceptor ligand is present in the protein nanoparticle and/or thestealth polymer-containing protein nanoparticle.

In certain embodiments, the protein comprises a plasma protein, hormone,immunomodulator, cytokine, interferon, interleukin, or enzyme. In otherembodiments, the protein comprises an antibody. In yet otherembodiments, the protein comprises an immunoglobulin. In yet otherembodiments, the immunoglobulin comprises IgA, IgD, IgE, IgG or IgM. Inyet other embodiments, the immunoglobulin comprises IgG.

In certain embodiments, the stealth polymer comprises an alkylpolyethylene oxide. In other embodiments, the stealth polymer comprisesan alkylphenol polyethylene oxide. In yet other embodiments, the stealthpolymer comprises a copolymer of polyethylene oxide and polypropyleneoxide. In yet other embodiments, the non-ionic surfactant comprises analkyl polyglucoside. In yet other embodiments, the non-ionic surfactantcomprises a fatty alcohol. In yet other embodiments, the non-ionicsurfactant comprises a cocamide MEA. In yet other embodiments, thenon-ionic surfactant comprises a cocamide DEA. In yet other embodiments,the stealth polymer comprises one or more of the polymers recitedelsewhere herein.

In certain embodiments, the alkyl polyethylene oxide comprisesdiethylene glycol hexadecyl ether. In other embodiments, the alkylpolyethylene oxide comprises polyethylene glycol oleyl ether. In yetother embodiments, the alkyl polyethylene oxide comprises diethyleneglycol octadecyl ether. In yet other embodiments, the alkyl polyethyleneoxide comprises polyoxyethylene stearyl ether. In yet other embodiments,the alkyl polyethylene oxide comprises polyethylene glycol hexadecyl(cetyl) ether. In yet other embodiments, the alkyl polyethylene oxidecomprises polyethylene glycol dodecyl (lauryl) ether. In yet otherembodiments, the alkyl polyethylene oxide comprises decaethylene glycololeyl ether. In yet other embodiments, the alkyl polyethylene oxidecomprises polyethylene glycol octadecyl ether. In yet other embodiments,the alkyl polyethylene oxide comprises polyethylene glycol octadecylether. In yet other embodiments, the alkyl polyethylene oxide comprisesone or more of the polymers recited elsewhere herein.

In certain embodiments, the average diameter of the at least one proteinnanoparticle ranges from about 1 nm to about 1,000 nm. In otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 10 nm to about 900 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 300 nm to about 600 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 250 nm to about 700 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 10 nm to about 120 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 20 nm to about 120 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 40 nm to about 120 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 60 nm to about 120 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 80 nm to about 120 nm. In yet otherembodiments, the average diameter of the at least one proteinnanoparticle ranges from about 100 nm to about 120 nm.

In certain embodiments, the concentration of the stealth polymer in thesolution ranges from about 0.1% to about 20,000% of the CMC of thestealth polymer. In other embodiments, the concentration of the stealthpolymer in the solution ranges from about 1% to about 100% of the CMC ofthe stealth polymer. In yet other embodiments, the concentration of thestealth polymer in the solution ranges from about 10% to about 100% ofthe CMC of the stealth polymer. In yet other embodiments, theconcentration of the stealth polymer in the solution ranges from about100% to about 20,000% of the CMC of the stealth polymer. In yet otherembodiments, the concentration of the stealth polymer in the solutionranges from about 300% to about 10,000% of the CMC of the stealthpolymer. In yet other embodiments, the concentration of the stealthpolymer in the solution ranges from about 300% to about 5,000% of theCMC of the stealth polymer. In yet other embodiments, the compositionfurther comprises a pharmaceutically acceptable carrier.

In certain embodiments of the present invention, the antibody comprisesa monoclonal antibody. In other embodiments, the monoclonal antibodycomprises bevacizumab, anatumomab, benralizumab, enokizumab, mitumomab,oxelumab, palivizumab or any combinations thereof.

Surfactants

Non-limiting examples of stealth polymers useful within the compositionsand methods of the present invention are alkyl polyethylene oxide (suchas, but not limited to, diethylene glycol hexadecyl ether, polyethyleneglycol oleyl ether, diethylene glycol octadecyl ether, polyoxyethylenestearyl ether, polyethylene glycol hexadecyl (cetyl) ether, polyethyleneglycol dodecyl (lauryl) ether, decaethylene glycol oleyl ether,polyethylene glycol octadecyl ether, and polyethylene glycol octadecylether), alkylphenol polyethylene oxide, copolymers of polyethylene oxideand polypropylene oxide (known as poloxamers or poloxamines), alkylpolyglucosides (including octyl glucoside and decyl maltoside), fattyalcohols (including cetyl alcohol and oleyl alcohol), cocamide MEA, andcocamide DEA.

Ligands

In certain embodiments, the nanoparticles of the present inventionfurther comprise at least one cell surface receptor ligand. In certainembodiments, the ligands allow for the nanoparticles of the presentinvention to recognize and bind to a cell that displays such cellsurface receptor.

Non-limiting examples of ligands contemplated within the inventioninclude ligands that bind to at least one of the following receptors:neurotensin receptor-1, human epidermal growth factor receptor-2(HER-2), folate receptor, insulin-like growth (IGF) receptor, and/orepidermal growth factor receptor (EGFR).

Non-limiting examples of ligands contemplated within the inventioninclude anti-NTSR1-mAb or SR-48692 (also known as2-[[[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)-1H-pyrazol-3-yl]carbonyl]amino]-tricyclo[3.3.1.13,7]decane-2-carboxylicacid), which bind to neurotensin receptor-1: trastuzumab, which binds toHER-2; folic acid (also known as(2S)-2-[[4-[(2-amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid,N-(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}benzoyl)-L-glutamicacid; pteroyl-L-glutamic acid; Vitamin B9; or folacin), which binds tothe folate receptor; anti-IGF-mAb (such as MK-0646, MA5-12247, AVE1642,figitumumab, or IMC-A12), which binds to the IGF receptor; andgefinitib, erlotinib, panitumumab, cetuximab, zalutumumab, nimotuzumabor matuzumab, which bind to the EGFR receptor.

Antibodies

Antibodies are useful within the compositions and methods of the presentinvention. In certain embodiments, the antibody comprises IgG,bevacizumab, anatumomab, benralizumab, enokizumab, mitumomab, oxelumab,palivizumab and any combinations thereof within the methods of thepresent invention. In other embodiments, the antibody is human orhumanized. In yet other embodiments, the antibody comprises an antibodyselected from a polyclonal antibody, a monoclonal antibody, a humanizedantibody, a synthetic antibody, a heavy chain antibody, a humanantibody, and a biologically active fragment of an antibody.

Non-limiting examples of antibodies useful within the compositions andmethods of the present invention include:

bevacizumab (AVASTIN®): humanized monoclonal antibody that inhibitsvascular endothelial growth factor A (VEGF-A) and is used to treatcancers such as colon cancer, rectum cancer, lung cancer, glioblastoma,and renal cell cancer (Los et al., 2007, The Oncologist 12(4):443-50);

anatumomab mafenatox: a mouse monoclonal antibody for the treatmentnon-small cell lung cancer; a fusion protein of a Fab fragment with anenterotoxin (“mafenatox”) of S. aureus;

benralizumab: a monoclonal antibody for the treatment of asthma, anddirected against the alpha-chain of the interleukin-5 receptor (CD125)(Catley, 2010, IDrugs: Invest. Drugs J. 13 (9):601-604);

enokizumab: a humanized monoclonal antibody designed for the treatmentof asthma;

mitumomab (BEC-2): a mouse monoclonal antibody investigated for thetreatment of small cell lung carcinoma;

oxelumab: human monoclonal antibody designed for the treatment ofasthma; and,

palivizumab (SYNAGIS®): a monoclonal antibody used in the prevention ofrespiratory syncytial virus (RSV) infections; a humanized monoclonalantibody (IgG) directed against an epitope in the A antigenic site ofthe F protein of RSV.

It will be appreciated by one skilled in the art that an antibodycomprises any immunoglobulin molecule, whether derived from naturalsources or from recombinant sources, which is able to specifically bindto an epitope present on a target molecule.

In one aspect of the present invention, the target molecule is directlyneutralized by an antibody that specifically binds to an epitope on thetarget molecule. In another aspect of the present invention, the effectsof the target molecule are blocked by an antibody that specificallybinds to an epitope on a downstream effector. In still another aspect ofthe present invention, the effects of the target molecule are blocked byan antibody that binds to an epitope of an upstream regulator of thetarget molecule.

When the antibody to the target molecule used in the compositions andmethods of the present invention is a polyclonal antibody (IgG), theantibody is generated by inoculating a suitable animal with a peptidecomprising full length target protein, or a fragment thereof, anupstream regulator, or fragments thereof. These polypeptides, orfragments thereof, may be obtained by any methods known in the art,including chemical synthesis and biological synthesis, as describedelsewhere herein. Antibodies produced in the inoculated animal thatspecifically bind to the target molecule, or fragments thereof, are thenisolated from fluid obtained from the animal.

Antibodies may be generated in this manner in several non-human mammalssuch as, but not limited to goat, sheep, horse, camel, rabbit, anddonkey. Methods for generating polyclonal antibodies are well known inthe art and are described, for example in Harlow et al., 1998, In:Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.

Monoclonal antibodies directed against a full length target molecule, orfragments thereof, may be prepared using any well-known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1998, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115). Humanmonoclonal antibodies may be prepared by the method described in U.S.Patent Publication No. 2003/0224490. Monoclonal antibodies directedagainst an antigen are generated from mice immunized with the antigenusing standard procedures as referenced herein. Nucleic acid encodingthe monoclonal antibody obtained using the procedures described hereinmay be cloned and sequenced using technology which is available in theart, and is described, for example, in Wright et al., 1992, CriticalRev. Immunol. 12(3,4):125-168, and the references cited therein.

When the antibody used in the methods of the present invention is abiologically active antibody fragment or a synthetic antibodycorresponding to antibody to a full length target molecule, or fragmentsthereof, the antibody is prepared as follows: a nucleic acid encodingthe desired antibody or fragment thereof is cloned into a suitablevector. The vector is transfected into cells suitable for the generationof large quantities of the antibody or fragment thereof. DNA encodingthe desired antibody is then expressed in the cell thereby producing theantibody. The nucleic acid encoding the desired peptide may be clonedand sequenced using technology available in the art, and described, forexample, in Wright et al., 1992, Critical Rev. in Immunol.12(3,4):125-168 and the references cited therein. Alternatively,quantities of the desired antibody or fragment thereof may also besynthesized using chemical synthesis technology. If the amino acidsequence of the antibody is known, the desired antibody can bechemically synthesized using methods known in the art as describedelsewhere herein.

The present invention also includes the use of humanized antibodiesspecifically reactive with an epitope present on a target molecule.These antibodies are capable of binding to the target molecule. Thehumanized antibodies useful in the invention have a human framework andhave one or more complementarity determining regions (CDRs) from anantibody, typically a mouse antibody, specifically reactive with atargeted cell surface molecule.

When the antibody used in the invention is humanized, the antibody canbe generated as described in Queen et al. (U.S. Pat. No. 6,180,370),Wright et al., 1992, Critical Rev. Immunol. 12(3,4):125-168, and in thereferences cited therein, or in Gu et al., 1997, Thrombosis & Hematocyst77(4):755-759, or using other methods of generating a humanized antibodyknown in the art. The method disclosed in Queen et al. is directed inpart toward designing humanized immunoglobulins that are produced byexpressing recombinant DNA segments encoding the heavy and light chaincomplementarity determining regions (CDRs) from a donor immunoglobulincapable of binding to a desired antigen, attached to DNA segmentsencoding acceptor human framework regions. Generally speaking, theinvention in the Queen patent has applicability toward the design ofsubstantially any humanized immunoglobulin. Queen explains that the DNAsegments typically include an expression control DNA sequence operablylinked to humanized immunoglobulin coding sequences, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells, or the expressioncontrol sequences can be prokaryotic promoter systems in vectors capableof transforming or transfecting prokaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the introducednucleotide sequences and as desired the collection and purification ofthe humanized light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments or other immunoglobulin forms mayfollow (Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NewYork, (1979), which is incorporated herein by reference).

Human constant region (CDR) DNA sequences from a variety of human cellscan be isolated in accordance with well-known procedures. Preferably,the human constant region DNA sequences are isolated from immortalizedB-cells as described in International Patent Application Publication No.WO 198702671. CDRs useful in producing the antibodies of the presentinvention may be similarly derived from DNA encoding monoclonalantibodies capable of binding to the target molecule. Such humanizedantibodies may be generated using well-known methods in any convenientmammalian source capable of producing antibodies, including, but notlimited to, mice, rats, camels, llamas, rabbits, or other vertebrates.Suitable cells for constant region and framework DNA sequences and hostcells in which the antibodies are expressed and secreted, can beobtained from a number of sources, such as the American Type CultureCollection, Manassas, Va.

One of skill in the art will further appreciate that the presentinvention encompasses the use of antibodies derived from camelidspecies. That is, the present invention includes, but is not limited to,the use of antibodies derived from species of the camelid family. As iswell known in the art, camelid antibodies differ from those of mostother mammals in that they lack a light chain, and thus comprise onlyheavy chains with complete and diverse antigen binding capabilities(Hamers-Casterman et al., 1993, Nature, 363:446-448). Such heavy-chainantibodies are useful in that they are smaller than conventionalmammalian antibodies, they are more soluble than conventionalantibodies, and further demonstrate an increased stability compared tosome other antibodies. Camelid species include, but are not limited toOld World camelids, such as two-humped camels (C. bactrianus) and onehumped camels (C. dromedarius). The camelid family further comprises NewWorld camelids including, but not limited to llamas, alpacas, vicuna andguanaco. The production of polyclonal sera from camelid species issubstantively similar to the production of polyclonal sera from otheranimals such as sheep, donkeys, goats, horses, mice, chickens, rats, andthe like. The skilled artisan, when equipped with the present disclosureand the methods detailed herein, can prepare high-titers of antibodiesfrom a camelid species. As an example, the production of antibodies inmammals is detailed in such references as Harlow et al., 1998,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.

V_(H) proteins isolated from other sources, such as animals with heavychain disease (Seligmann et al., 1979, Immunological Rev. 48:145-167,incorporated herein by reference in its entirety), are also useful inthe compositions and methods of the present invention. The presentinvention further comprises variable heavy chain immunoglobulinsproduced from mice and other mammals, as detailed in Ward et al., 1989,Nature 341:544-546 (incorporated herein by reference in its entirety).Briefly, V_(H) genes are isolated from mouse splenic preparations andexpressed in E. coli. The present invention encompasses the use of suchheavy chain immunoglobulins in the compositions and methods detailedherein.

Antibodies useful as target molecule depletors in the invention may alsobe obtained from phage antibody libraries. To generate a phage antibodylibrary, a cDNA library is first obtained from mRNA which is isolatedfrom cells, e.g., the hybridoma, which express the desired protein to beexpressed on the phage surface, e.g., the desired antibody. cDNA copiesof the mRNA are produced using reverse transcriptase. cDNA thatspecifies immunoglobulin fragments are obtained by PCR and the resultingDNA is cloned into a suitable bacteriophage vector to generate abacteriophage DNA library comprising DNA specifying immunoglobulingenes. The procedures for making a bacteriophage library comprisingheterologous DNA are well known in the art and are described, forexample, in Sambrook et al., 2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Bacteriophage that encode the desired antibody may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage that express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage that do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al., 1992,Critical Rev. Immunol. 12(3,4):125-168.

Processes such as those described above have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage that display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage thatencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al., 1991, J. Mol. Biol. 222:581-597. Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.,1995, J. Mol. Biol. 248:97-105).

Once expressed, whole antibodies, dimers derived therefrom, individuallight and heavy chains, or other forms of antibodies can be purifiedaccording to standard procedures known in the art. Such proceduresinclude, but are not limited to, ammonium sulfate precipitation, the useof affinity columns, routine column chromatography, gel electrophoresis,and the like (see, generally, R. Scopes, “Protein Purification”,Springer-Verlag, N.Y. (1982)). Substantially pure antibodies of at leastabout 90% to 95% homogeneity are contemplated; and antibodies having 98%to 99% or more homogeneity are also contemplated for pharmaceuticaluses. Once purified, the antibodies may then be used to practice themethod of the present invention, or to prepare a pharmaceuticalcomposition useful in practicing the method of the present invention.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassaysthat can be used include but are not limited to competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g, CurrentProtocols in Molecular Biology, (Ausubel et al., eds.), GreenePublishing Associates and Wiley-Interscience, New York (2002)).Exemplary immunoassays are described briefly below (but are not intendedto be in any way limiting).

Therapeutic Agents

In certain embodiments, the nanoparticles of the present inventionfurther comprise at least one therapeutic agent. The at least onetherapeutic agent may be a therapeutic, prophylactic, and/or diagnosticagent. Any suitable therapeutic agent may be used within thecompositions and methods of the present invention. Non-limiting examplesof therapeutic agent contemplated within the invention include organiccompounds, inorganic compounds, antibodies (such as any of theantibodies discussed elsewhere herein), hydrophobic or hydrophilicpharmacological drugs, radiopharmaceuticals, biologics, proteins,peptides, polysaccharides, nucleic acids, siRNA, miRNA, RNAi, shorthairpin RNAs (shRNAs), antisense nucleic acids), ribozymes, dominantnegative mutants, or other materials that can be incorporated into thenanoparticles using standard techniques and/or the methods describedherein.

In certain embodiments, the nanoparticles comprise an interfering RNAthat reduces translation of at least one cell protein and/or polypeptidein a cell of a subject, wherein the cell protein is associated with adisease or disorder in the subject. An interfering RNA can include asiRNA, a shRNA, and a microRNA. An siRNA polynucleotide is an RNAnucleic acid molecule that interferes with RNA activity that isgenerally considered to occur via a post-transcriptional gene silencingmechanism. An siRNA polynucleotide preferably comprises adouble-stranded RNA (dsRNA) but is not intended to be so limited and maycomprise a single-stranded RNA (see, e.g., Martinez et al., 2002 Cell110:563-74). The siRNA polynucleotide included in the invention maycomprise other naturally occurring, recombinant, or syntheticsingle-stranded or double-stranded polymers of nucleotides(ribonucleotides or deoxyribonucleotides or a combination of both)and/or nucleotide analogues as provided herein (e.g., an oligonucleotideor polynucleotide or the like, typically in 5′- to 3′-phosphodiesterlinkage). Accordingly, it will be appreciated that certain exemplarysequences disclosed herein as DNA sequences capable of directing thetranscription of the siRNA polynucleotides are also intended to describethe corresponding RNA sequences and their complements, given thewell-established principles of complementary nucleotide base-pairing.

Contemplated siRNA polynucleotides comprise double-strandedpolynucleotides of about 18-30 nucleotide base pairs, for example about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, or about 27 base pairs, and in other embodiments about 19,about 20, about 21, about 22 or about 23 base pairs, or about 27 basepairs, whereby the use of “about” indicates that in certain embodimentsand under certain conditions the processive cleavage steps that may giverise to functional siRNA polynucleotides that are capable of interferingwith expression of a selected polypeptide may not be absolutelyefficient. Hence, siRNA polynucleotides may include one or more siRNApolynucleotide molecules that may differ (e.g., by nucleotide insertionor deletion) in length by one, two, three, four or more base pairs as aconsequence of the variability in processing, in biosynthesis, or inartificial synthesis of the siRNA. The siRNA polynucleotide of thepresent invention may also comprise a polynucleotide sequence thatexhibits variability by differing (e.g., by nucleotide substitution,including transition or transversion) at one, two, three or fournucleotides from a particular sequence. These differences can occur atany of the nucleotide positions of a particular siRNA polynucleotidesequence, depending on the length of the molecule, whether situated in asense or in an antisense strand of the double-stranded polynucleotide.The nucleotide difference may be found on one strand of adouble-stranded polynucleotide, where the complementary nucleotide withwhich the substitute nucleotide would typically form hydrogen bond basepairing, may not necessarily be correspondingly substituted. In certainembodiments, the siRNA polynucleotides are homogeneous with respect to aspecific nucleotide sequence.

It should be appreciated that the siRNAs of the present invention mayeffect silencing of the target polypeptide expression to differentdegrees. Selection of siRNAs are made therefrom based on the ability ofa given siRNA to interfere with or modulate the expression of the targetpolypeptide. The methods for testing each siRNA and selection ofsuitable siRNAs for use in the present invention are fully known tothose skilled in the art. It is appreciated by one skilled in the artthat siRNAs are easily designed and manufactured. Further, effects ofsiRNA are typically transient in nature, which make them optimal forcertain therapies where sustained inhibition is undesired. Another formof an interfering RNA, shRNA polynucleotides utilize the endogenousprocessing machinery of the cell and are often designed for highpotency, sustainable effects, and fewer off-target effects (Rao et al.,2009, Adv Drug Deliv Rev, 61: 746-759). As would be understood by thoseskilled in the art, the present invention encompasses both siRNA andshRNA polynucleotides, which can be designed and delivered to inhibitone or more cell proteins.

One skilled in the art will appreciate that one way to decrease the mRNAand/or protein levels of a cell protein is by reducing or inhibitingexpression of the nucleic acid encoding the cell protein. Thus, thelevel of the cell protein in a cell can also be decreased using amolecule or compound that inhibits or reduces gene expression such as,for example, an antisense molecule or a ribozyme.

In certain embodiments, the modulating sequence is an antisense nucleicacid sequence expressed by a plasmid vector. The antisense expressingvector is used to transfect a mammalian cell or the mammal itself,thereby causing reduced endogenous expression of a desired protein inthe cell. However, the invention should not be construed to be limitedto inhibiting expression of a protein by transfection of cells withantisense molecules. Rather, the invention encompasses other methodsknown in the art for inhibiting expression or activity of a protein inthe cell including, but not limited to, the use of a ribozyme, theexpression of a non-functional protein (i.e. dominant negative mutant)and use of an intracellular antibody.

Antisense molecules and their use for inhibiting gene expression arewell known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

Ribozymes and their use for inhibiting gene expression are also wellknown in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are RNA molecules possessingthe ability to specifically cleave other single-stranded RNA in a manneranalogous to DNA restriction endonucleases. Through the modification ofnucleotide sequences encoding these RNAs, molecules can be engineered torecognize specific nucleotide sequences in an RNA molecule and cleave it(Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of thisapproach is the fact that ribozymes are sequence-specific.

In another aspect of the present invention, the protein can be inhibitedby way of inactivation and/or sequestration. As such, inhibiting theeffects of a protein can be accomplished by using a dominant negativemutant. Alternatively an antibody specific for the desired protein,otherwise known as an antagonist to the protein, may be used. In certainembodiments, the antagonist is a protein and/or compound having thedesirable property of interacting with a binding partner of the proteinand thereby competing with the corresponding wild-type protein. Inanother embodiments, the antagonist is a protein and/or compound havingthe desirable property of interacting with the protein and therebysequestering the protein.

Inhibition of one or more cell proteins can be accomplished using amodified nucleic acid molecule, such as a small interfering RNA (siRNA),short hairpin RNA (shRNA), a microRNA, an antisense nucleic acid, aribozyme, an expression vector encoding a dominant negative mutant, andthe likes. The methods of modifying nucleic acid molecules are known inthe art. For example, a number of specific siRNA polynucleotidesequences useful for interfering with target polypeptide expression areknown in the art. siRNA polynucleotides may generally be prepared by anymethod known in the art, including, for example, solid phase chemicalsynthesis. Modifications in a polynucleotide sequence may also beintroduced using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis. Further, siRNAs maybe chemically modified or conjugated with other molecules to improvetheir stability and/or delivery properties. Included as one aspect ofthe present invention are siRNAs as described herein, wherein one ormore ribose sugars has been removed therefrom.

Alternatively, siRNA polynucleotide molecules may be generated by invitro or in vivo transcription of suitable DNA sequences (e.g.,polynucleotide sequences encoding a target polypeptide, or a desiredportion thereof), provided that the DNA is incorporated into a vectorwith a suitable RNA polymerase promoter (such as for example, T7, U6,H1, or SP6 although other promoters may be equally useful). In addition,an siRNA polynucleotide may be administered to a mammal, as may be a DNAsequence (e.g., a recombinant nucleic acid construct as provided herein)that supports transcription (and optionally appropriate processingsteps) such that a desired siRNA is generated in vivo.

In certain embodiments, an siRNA polynucleotide, wherein the siRNApolynucleotide is capable of interfering with expression of a targetpolypeptide can be used to generate a silenced cell. Any siRNApolynucleotide that, when contacted with a biological source for aperiod of time, results in a significant decrease in the expression ofthe target polypeptide is included in the invention. Preferably thedecrease is greater than about 10%, more preferably greater than about20%, more preferably greater than about 30%, more preferably greaterthan about 40%, about 50%, about 60%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 98% relative to the expressionlevel of the target polypeptide detected in the absence of the siRNA.Preferably, the presence of the siRNA polynucleotide in a cell does notresult in or cause any undesired toxic effects, for example, apoptosisor death of a cell in which apoptosis is not a desired effect of RNAinterference.

Any polynucleotide of the present invention may be further modified toincrease its stability in vivo. Possible modifications include, but arenot limited to, the addition of flanking sequences at the 5′ and/or 3′ends; the use of phosphorothioate or 2′-O-methyl rather thanphosphodiester linkages in the backbone; and/or the inclusion ofnon-traditional bases such as inosine, queosine, and wybutosine and thelike, as well as acetyl- methyl-, thio- and other modified forms ofadenine, cytidine, guanine, thymine, and uridine.

Methods

The invention provides a method of preparing at least one stealthpolymer-containing protein nanoparticle. In certain embodiments, themethod comprises adjusting the pH of a first solution comprising aprotein to about the isoelectric point of the protein, thereby forming afirst protein nanoparticle, which comprises at least a fraction of theprotein. In other embodiments, if the protein in the first proteinnanoparticle is not conjugated to at least one cell surface receptorligand, the protein in the first protein nanoparticle is furtherconjugated with the at least one cell surface receptor. In yet otherembodiments, the method comprises contacting the first proteinnanoparticle with a second solution comprising a stealth polymer,wherein the concentration of the stealth polymer in the second solutionranges from about 0.1% to about 20,000% of the CMC of the stealthpolymer. In yet other embodiments, the at least one stealthpolymer-containing protein nanoparticle is prepared.

The invention further provides a method of treating, ameliorating orpreventing a disease or disorder in a subject in need thereof. Incertain embodiments, the method comprises administering to the subject apharmaceutically effective amount of a composition of the invention.

In certain embodiments, the at least one stealth polymer-containingprotein nanoparticle is further purified to remove protein or stealthpolymer that is not associated with the at least one stealthpolymer-containing protein nanoparticle, thereby generating acomposition comprising the at least one stealth polymer-containingprotein nanoparticle.

In certain embodiments, the composition comprising at least one stealthpolymer-containing protein nanoparticle is further lyophilized.

In certain embodiments, the protein comprises at least one selected fromthe group consisting of a plasma protein, hormone, immunomodulator,cytokine, interferon, interleukin, and enzyme. In other embodiments, theprotein comprises an antibody. In yet other embodiments, the antibodycomprises IgA, IgD, IgE, IgG, and/or IgM.

In certain embodiments, the first solution further comprises at leastone therapeutic agent, and wherein the first protein nanoparticlecomprises at least a fraction of the at least one therapeutic agent. Inother embodiments, the at least therapeutic agent is selected from thegroup consisting of an organic compound, inorganic compound, antibody,pharmacological drug, radiopharmaceutical, protein, peptide,polysaccharide, nucleic acid, siRNA, RNAi, short hairpin RNA, antisensenucleic acid, ribozyme and dominant negative mutant. In yet otherembodiments, the at least therapeutic agent comprises a siRNA or miRNA.

In certain embodiments, the protein in the protein nanoparticle isconjugated non-covalently and/or covalently to the at least one cellsurface receptor ligand. In other embodiments, the at least one cellsurface receptor ligand binds to at least one selected from the groupconsisting of neurotensin receptor-1, human epidermal growth factorreceptor-2 (HER-2), folate receptor, insulin-like growth receptor (IGF),and epidermal growth factor receptor (EGFR). In yet other embodiments,the stealth polymer comprises at least one selected from the groupconsisting of alkyl polyethylene oxide, alkylphenol polyethylene oxide,copolymer of polyethylene oxide and polypropylene oxide, alkylpolyglucoside, fatty alcohol, cocamide MEA, and cocamide DEA. In yetother embodiments, the alkyl polyethylene oxide comprises at least oneselected from the group consisting of diethylene glycol hexadecyl ether,polyethylene glycol oleyl ether, diethylene glycol octadecyl ether,polyoxyethylene stearyl ether, polyethylene glycol hexadecyl (cetyl)ether, polyethylene glycol dodecyl (lauryl) ether, decaethylene glycololeyl ether, polyethylene glycol octadecyl ether, and polyethyleneglycol octadecyl ether.

In certain embodiments, the average diameter of the at least one stealthpolymer-containing nanoparticle ranges from about 1 nm to about 1,000nm. In other embodiments, the concentration of the stealth polymer inthe second solution ranges from about 100% to about 20,000% of the CMCof the stealth polymer. In yet other embodiments, the at least onestealth polymer-containing protein nanoparticle is formulated with apharmaceutically acceptable carrier.

In certain embodiments, the composition is administered to the subjectby an intrapulmonary, intrabronchial, inhalational, intranasal,intratracheal, intravenous, intramuscular, subcutaneous, topical,transdermal, oral, buccal, rectal, pleural, peritoneal, vaginal,epidural, otic, intraocular, or intrathecal route. In other embodiments,the composition is administered to the subject by an intrapulmonary,intrabronchial, inhalational, intranasal, intratracheal, intravenous,intramuscular, subcutaneous or topical route.

In certain embodiments, the composition further comprises apharmaceutically acceptable carrier.

In certain embodiments, the protein comprises IgG and the nanoparticlefurther comprises a therapeutic agent comprising a siRNA or a miRNA.

In certain embodiments, the stealth polymer comprises a copolymer ofpolyethylene oxide and polypropylene oxide.

In certain embodiments, the disease or disorder is selected from thegroup consisting of colon cancer, rectum cancer, lung cancer,glioblastoma, renal cell cancer, non-small cell lung cancer, small celllung cancer, asthma, respiratory syncytial virus (RSV) infection, andany combinations thereof. In other embodiments, the disease or disordercomprises a cancer comprising a KRAS mutation.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is human.

Pharmaceutical Compositions and Formulations

The invention also encompasses the use of pharmaceutical compositions ofat least one composition of the present invention or a salt thereof topractice the methods of the present invention.

Such a pharmaceutical composition may consist of at least onecomposition of the present invention or a salt thereof, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise at least one composition of the presentinvention or a salt thereof, and one or more pharmaceutically acceptablecarriers, one or more additional ingredients, or some combination ofthese. The at least one composition of the present invention may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

In certain embodiments, the pharmaceutical compositions useful forpracticing the method of the present invention may be administered todeliver a API dose of between 1 ng/kg/day and 100 mg/kg/day, between 1ng/kg/day and 500 mg/kg/day, or between 1 pg/kg/day and 10 ng/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the present invention will vary, depending upon theidentity, size, and condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.

Pharmaceutical compositions that are useful in the methods of thepresent invention may be suitably developed for inhalational, pulmonary,intranasal, intratracheal, intravenous, intramuscular, subcutaneous,topical, or another route of administration. Other contemplatedformulations include projected nanoparticles, containing the activeingredient, and immunologically-based formulations. The route(s) ofadministration are readily apparent to the skilled artisan and dependupon any number of factors including the type and severity of thedisease being treated, the type and age of the veterinary or humanpatient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose. The unit dosage form mayalso be for extended duration administration, such as once weekly oronce monthly, depending on the efficacy of the protein formulation andthe disease.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the present invention is contemplated include, but arenot limited to, humans and other primates, mammals includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and dogs.

In certain embodiments, the compositions of the present invention areformulated using one or more pharmaceutically acceptable excipients orcarriers. In certain embodiments, the pharmaceutical compositions of thepresent invention comprise a therapeutically effective amount of atleast one composition of the present invention and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers, which areuseful, include, but are not limited to, glycerol, water, saline,ethanol and other pharmaceutically acceptable salt solutions such asphosphates and salts of organic acids. Examples of these and otherpharmaceutically acceptable carriers are described in Remington'sPharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofstealth polymers. Prevention of the action of microorganisms may beachieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it is preferable to include isotonic agents, forexample, sugars, sodium chloride, or polyalcohols such as mannitol andsorbitol, in the composition.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for nasal, inhalational, or any other suitable modeof administration, known to the art. The pharmaceutical preparations maybe sterilized and if desired mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure buffers, coloring, flavoringand/or aromatic substances and the like. They may also be combined wheredesired with other active agents, e.g., other analgesic agents. As usedherein, “additional ingredients” include, but are not limited to, one ormore ingredients that may be used as a pharmaceutical carrier.

The composition of the present invention may comprise a preservativefrom about 0.005% to 2.0% by total weight of the composition. Thepreservative is used to prevent spoilage in the case of exposure tocontaminants in the environment. Examples of preservatives useful inaccordance with the invention included but are not limited to thoseselected from the group consisting of benzyl alcohol, sorbic acid,parabens, and combinations thereof. A contemplated preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an antioxidant and a chelating agentthat inhibit the degradation of the compound. Contemplated antioxidantsfor some compounds are butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), alpha-tocopherol (vitamin E) and ascorbic acid inthe range of about 0.01% to 0.3% and more preferably BHT in the range of0.03% to 0.1% by weight by total weight of the composition. Preferably,the chelating agent is present in an amount of from 0.01% to 0.5% byweight by total weight of the composition. Contemplated chelating agentsinclude edetate salts (e.g. disodium ethylenediaminetetracetic acid(EDTA)) and citric acid in the weight range of about 0.01% to 0.20% andmore preferably in the range of 0.02% to 0.10% by weight by total weightof the composition. The chelating agent is useful for chelating metalions in the composition which may be detrimental to the shelf life ofthe formulation. While BHT and disodium edetate are contemplatedantioxidant and chelating agent respectively for some compounds, othersuitable and equivalent antioxidants and chelating agents may besubstituted therefore as would be known to those skilled in the art.

Known suspending agents include, but are not limited to, sorbitol syrup,hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gumtragacanth, gum acacia, and cellulose derivatives such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.Known dispersing or wetting agents include, but are not limited to,naturally-occurring phosphatides such as lecithin, condensation productsof an alkylene oxide with a fatty acid, with a long chain aliphaticalcohol, with a partial ester derived from a fatty acid and a hexitol,or with a partial ester derived from a fatty acid and a hexitolanhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol,polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitanmonooleate, respectively). Known emulsifying agents include, but are notlimited to, lecithin, and acacia. Known preservatives include, but arenot limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates,ascorbic acid, and sorbic acid. Known sweetening agents include, forexample, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.Known thickening agents for oily suspensions include, for example,beeswax, hard paraffin, and cetyl alcohol.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after the onset of a disease or disorder. Further,several divided dosages, as well as staggered dosages may beadministered daily or sequentially, or the dose may be continuouslyinfused, or may be a bolus injection. Further, the dosages of thetherapeutic formulations may be proportionally increased or decreased asindicated by the exigencies of the therapeutic or prophylacticsituation.

When used in vivo, the compositions of the present invention arepreferably administered as a pharmaceutical composition, comprising amixture, and a pharmaceutically acceptable carrier. The compositions ofthe present invention may be present in a pharmaceutical composition inan amount from 0.001 to 99.9 wt %, more preferably from about 0.01 to 99wt %, and even more preferably from 0.1 to 95 wt %.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder in the patient. An effective amount ofthe therapeutic compound necessary to achieve a therapeutic effect mayvary according to factors such as the activity of the particularcomposition employed; the time of administration; the rate of excretionof the composition; the duration of the treatment; other drugs,compounds or materials used in combination with the composition; thestate of the disease or disorder, age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well-known in the medical arts. Dosage regimens may be adjustedto provide the optimum therapeutic response. For example, severaldivided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the present invention is from about 0.01 μg/kgand 10 mg/kg of body weight/per day. One of ordinary skill in the artwould be able to study the relevant factors and make the determinationregarding the effective amount of the therapeutic composition withoutundue experimentation.

The composition can be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. It is understood that the amount of composition dosed per daymay be administered, in non-limiting examples, every day, every otherday, every 2 days, every 3 days, every 4 days, or every 5 days. Forexample, with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the present inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the present invention are dictated by and directly dependent on(a) the unique characteristics of the therapeutic composition and theparticular therapeutic effect to be achieved, and (b) the limitationsinherent in the art of compounding/formulating such a therapeuticcomposition for the treatment of diseases or disorders in a patient.

In certain embodiments, the compositions of the present invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the presentinvention are administered to the patient in range of dosages thatinclude, but are not limited to, once every day, every two, days, everythree days to once a week, and once every two weeks. It will be readilyapparent to one skilled in the art that the frequency of administrationof the various combination compositions of the present invention willvary from subject to subject depending on many factors including, butnot limited to, age, disease or disorder to be treated, gender, overallhealth, and other factors. Thus, the invention should not be construedto be limited to any particular dosage regime and the precise dosage andcomposition to be administered to any patient will be determined by theattending physician taking all other factors about the patient intoaccount.

Compositions of the present invention for administration may be in therange of from about 1 μg to about 1,000 mg, about 2 μg to about 500 mg,about 4 μg to about 250 mg, about 6 μg to about 200 mg, about 8 μg toabout 100 mg, about 10 μg to about 50 mg, about 20 μg to about 25 mg,about 40 μg to about 10 mg, about 50 μg to about 5 mg, about 100 μg toabout 1 mg, and any and all whole or partial increments thereinbetween.

In some embodiments, the dose of a composition of the present inventionis from about 0.5 μg and about 2,000 mg. In some embodiments, a dose ofa composition described herein is less than about 2,000 mg, or less thanabout 1,000 mg, or less than about 500 mg, or less than about 250 mg, orless than about 100 mg, or less than about 50 mg, or less than about 25mg, or less than about 10 mg, or less than about 5 mg, or less thanabout 1 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a composition of the presentinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the composition to treat, prevent, or reduceone or more symptoms of a disorder or disease in a patient.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in certain embodiments, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating, preventing, orreducing a breathing disorder in a patient.

Routes of Administration

Routes of administration of any of the compositions of the presentinvention include intrapulmonary, intrabronchial, inhalational,intranasal, intratracheal, intravenous, intramuscular, subcutaneous,topical, transdermal, oral, buccal, rectal, pleural, peritoneal,vaginal, epidural, otic, intraocular, or intrathecal administration.

Suitable compositions and dosage forms include, for example,suspensions, granules, beads, powders, pellets, and liquid sprays fornasal administration, dry powder or aerosolized formulations forinhalation, and the like. It should be understood that the formulationsand compositions that would be useful in the present invention are notlimited to the particular formulations and compositions that aredescribed herein. For example, formulations may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations mayfurther comprise one or more of the additional ingredients describedherein. The examples of formulations described herein are not exhaustiveand it is understood that the invention includes additionalmodifications of these and other formulations not described herein, butwhich are known to those of skill in the art.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention canbe, but are not limited to, short-term release or rapid-offset release,as well as controlled release, for example, sustained release, delayedrelease and pulsatile release formulations.

The term short-term or rapid-offset release is used in its conventionalsense to refer to a drug formulation that provides for release of thedrug immediately after drug administration.

As used herein, short-term or rapid-offset refers to any period of timeup to and including about 8 hours, about 7 hours, about 6 hours, about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about40 minutes, about 20 minutes, or about 10 minutes and any or all wholeor partial increments there between after drug administration after drugadministration.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time can be as long as a month or more and shouldbe longer than the time required for the release of the same amount ofagent administered in bolus form.

For sustained release, the compounds can be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds of the presentinvention can be administered in the form of microparticles for example,by injection or in the form of wafers or discs by implantation.

In certain embodiments of the present invention, the compositions of thepresent invention are administered to a subject, alone or in combinationwith another pharmaceutical agent, using a sustained releaseformulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, include a delay of from about 10 minutes up toabout 12 hours.

In certain embodiments of the present invention, the compositions of thepresent invention are administered to a subject, alone or in combinationwith another pharmaceutical agent, using a delayed release formulation.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

In certain embodiments of the present invention, the compositions of thepresent invention are administered to a subject, alone or in combinationwith another pharmaceutical agent, using a pulsatile releaseformulation.

Kits

The invention also includes a kit comprising a composition of thepresent invention and an instructional material that describesadministering the composition to a mammal. As used herein, an“instructional material” includes a publication, a recording, a diagram,or any other medium of expression that can be used to communicate theusefulness of the composition of the present invention in the kit foreffecting alleviation of the various diseases or disorders recitedherein.

Optionally, or alternatively, the instructional material may describeone or more methods of alleviating the diseases or disorders in a cellor a tissue of a mammal. The instructional material of the kit may, forexample, be affixed to a container that contains the invention or beshipped together with a container that contains the invention.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thecompound be used cooperatively by the recipient.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application. The following examples further illustrate aspectsof the present invention. However, they are in no way a limitation ofthe teachings or disclosure of the present invention as set forthherein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Experimental Details Materials

Human IgG was purchased from Equitech-Bio Inc. (Kerrville, Tex., USA).Poloxamer-188, RNase-free water, 4,6-diamidino-2-phenylindole (DAPI),HCl, and fetal bovine serum (FBS) were obtained from Thermo FisherScientific (Waltham, Mass., USA).

Aptamer against MUC1 (5′-GCA GTT GAT CCT TTG GAT ACC CTG G-3′; SEQ IDNO:1) was designed and purchased from GE Healthcare Bio-Sciences Corp.(Piscataway, N.J., USA). A C12 spacer was attached to the aptamer at the3′-end to form the following sequence: 5′-GCA GTT GAT CCT TTG GAT ACCCTG G-C₁₂H₂₅-3′ (SEQ ID NO:1—C₁₂H₂₅). One aptamer was modified with3′—NH₂ and 5′-FITC, whereas the other was only modified with 3′-NH₂. Theaptamers contained a 12-carbon spacer.

siGLO-Green (6-FAM-labeled) was obtained from GE Healthcare Bio-SciencesCorp. HCl was obtained from Thermo Fisher Scientific. MiRIDIAN mimicwith miR-29b and miRIDIAN mimic NC were obtained from GE HealthcareBio-Sciences Corp. Pierce RIPA lysis buffer was purchased from ThermoFisher Scientific. Rabbit antihuman DNMT3B and rabbit antihuman MCL1antibody were obtained from Thermo Fisher Scientific. Mouse antihumanβ-tubulin antibody was purchased from Sigma-Aldrich (St Louis, Mo.,USA).

Cell Culture

Adenocarcinoma cell line A549 and normal lung fibroblast cell line MRC-5were obtained from American Type Culture Collection (Rockville, Md.,USA). A549 cell was originally derived from a 58-year-old maleCaucasian, while MRC-5 was originally derived from a 14-week gestationmale Caucasian. A549 cells were maintained in F12 K medium supplementedwith 10% FBS and 1% antibiotics. MRC-5 was maintained in Eagle's MinimumEssential Medium supplemented with 10% FBS. Both cells were kept in ahumidified air atmosphere with 5% carbon dioxide.

Preparation of Nanoparticles

Human IgG was diluted in 0.01N HCl to make up IgG concentration of 1mg/mL (i.e., 10 mg) in 10 mL of 0.01 N HCl. A total of 0.73 mg ofmiRNA-29b was then added. The mixture was stirred on a magnetic stirreruntil all the components were fully dissolved. The mixture was titratedwith 0.01 N NaOH up to a pH value close to 7. Nanoparticles werespontaneously formed at the pH value very close to 7. The nanoparticleswere allowed to mix on the magnetic stirrer for about 10 minutes. Thecolloidal suspension was centrifuged using a microcentrifuge (Eppendorfcentrifuge 5418) at 2,000 rpm for 5 minutes. The supernatant was eitherdecanted or kept for the measurement of unencapsulated miRNA-29b to beused to calculate encapsulation efficiency and loading capacity.Nanoparticles were then rinsed three times with double distilleddeionized water. Nanoparticles were subsequently suspended in 0.2% v/vpoloxamer-188, with gentle shaking for 10 minutes in order to coatnanoparticles with poloxamer-188. Nanoparticles were centrifuged and thesupernatant decanted before being rinsed thrice with double distilleddeionized water. Particles were then loaded into a freeze dryer(Labconco Freeze Zone 4.6), and lyophilization was performed for 48hours.

Conjugation of Aptamer to Prepared Nanoparticles

A total of 50 μL of poloxamer-free nanoparticle suspension (10 μg/mL inDNase, RNase-free water) was mixed with 100 μL of 40 mM EDC and 100 μLof 10 mM NHS for 15 minutes at room temperature with gentle stirring.This helps to activate the carboxyl groups on nanoparticles for aptamerconjugation. A total of 50 μL of 1 μg/mL aptamer in DNase, RNAse-freewater was then added to the nanoparticles and mixed gently for 2 hoursat room temperature. Nanoparticles were subsequently centrifuged at4,000 rpm and 10° C. for 5 minutes using 30 kDa cutoff centrifugalultrafilters (EMD Millipore, Billerica, Mass., USA) to exclude unreactedEDC and NHS. Aptamer-functionalized nanoparticles were then suspended in0.2% v/v poloxamer-188, with gentle shaking for 10 minutes in order tocoat them with poloxamer-188. FIG. 6 illustrates a non-limiting sequenceinvolved in the preparation of MAFMILHN.

Characterization of Nanoparticles

Particle size and surface charge (zeta potential) of the nanoparticleswere measured using photon correlation spectroscopy (ZetaSizer Nano ZS;Malvern Instruments, Malvern, UK). Nanoparticles were dispersed indeionized water followed by sonication for about 5 minutes. A scatteringangle (0) of 173° was used to measure intensity autocorrelation. TheZ-average and polydispersity index were recorded in triplicate. For zetapotential, samples were taken in a universal dip cell (MalvernInstruments) and the zeta potential recorded in triplicate.

The shape of nanoparticles was evaluated using scanning electronmicroscopy. Suspensions of nanoparticles were dropped on an aluminumstub and allowed to dry at room temperature. Samples were then coatedwith a thin layer of palladium. Coated samples were imaged using a ZeissSupra 50 V system (Carl Zeiss Meditec AG, Jena, Germany).

Fourier Transform-Infrared Spectroscopy

A single-reflection-attenuated total reflectance with a diamond internalreflection crystal installed in an iS10 FT-IR spectrometer (ThermoFisher Scientific) was used to obtain spectra from different samples ofnanoparticles. Lyophilized powders were placed on the surface of theattenuated total reflectance crystal after background spectra had beencollected. Spectra were collected after 64 scans at 4 cm-1 resolution.Data were analyzed using OMNIC software.

miRNA Release Study

Release of miRNA from MUC1 aptamer-functionalized hybrid nanoparticleswas measured at pH values 5, 6.6, and 7.4. Nanoparticles (3 mg) weredispersed in 0.5 mL of a buffered solution in a tubular cellulosedialysis membrane secured tightly at both ends. This was then incubatedat 37° C. in 5 mL buffered solution reservoirs while the reservoir wasgently agitated. The amount of miRNA released at different time pointswas analyzed and quantified for percentage cumulative release usingion-pair HPLC.

Ion-Pair HPLC

miRNA was quantitatively determined using a Waters 2695 separationmodule combined with a Waters 2998 photodiode array detector AllianceHPLC system (Waters, Milford, Mass., USA). Using a Clarity 3 μm Oligo-RPcolumn (Phenomenex, Torrance, Calif., USA) with a column dimension of50×2.0 mm, 1 μL of miRNA sample was injected into the HPLC using 20 mMtriethylamine-acetic acid (pH 7) and 5%-12% acetonitrile, gradientelusion as mobile phase. A flow rate of 0.2 mL/min was used foranalysis. UV detection was performed at 269 nm, and Empower Pro softwarewas used to record chromatograms.

Isolation of miRNA-29b and Ion-Pair HPLC

Using Clarity OTX kit (Phenomenenex, CA) with Thermo Scientific HyperSepVacuum Manifold (ThermoScientific, Rockwood, Tenn.), miRNA-29b wasisolated from blood and tissues for ion-pair HPLC analysis according tomanufacturer's instructions. Equal aliquots of Clarity OTX loadingbuffer was mixed with plasma samples which were then loaded on the solidPhase Extraction (SPE) cartridge. Equilibration of the SPE isolationcartridge (Clarity OTX 100 mg/3 mL: Phenomenex) was done after it wasfirst wetted with methanol. Equilibration was done with Clarityequilibration buffer (10 mM phosphate pH 5.5). To isolate miRNA-29b fromtissue samples, tissues were homogenized in 0.1 M Tris buffer, pH 8.0and then mixed with equal amount of Lysis-loading buffer. Followingsample loading, equilibration buffer was added to the cartridge to rinsetwice. The cartridge was then rinsed with Clarity OTX wash buffer (10 mMphosphate pH 5.5/50% acetonitrile). MiRNA-29b was then eluted with 100mM ammonium bicarbonate pH 8.0/40% acetonitrile/10% tetrahydrofuran.Samples were then concentrated to 100 μL with speed vacuum beforeion-pair HPLC analysis.

Fluorescence Microscopy

A549 cells were seeded at a density of 2×10⁴ in eight-well coated glassslides (Discovery Labware, Tewksbury, Mass., USA) followed by incubationfor 48 hours. Cells were washed with PBS and incubated with eitherFITC-MUC1 aptamer-functionalized miRNA-29b-loaded hybrid nanoparticlesor MUC1 aptamer-functionalized siGLO-FAM-loaded hybrid nanoparticlesdispersed in the Opti-Mem medium at a concentration of 100 m/mL for 2hours or 4 hours. Cells were then washed twice with PBS, fixed using 2%paraformaldehyde, and incubated at room temperature for 20 minutes.Cells washed with PBS were then blocked with 5% BSA for 30 minutes atroom temperature. Cells were stained with either Lysotracker-Red orAlexaFluoro-555-labeled wheat germ agglutinin (WGA-AF-555) before beingstained with DAPI to visualize nucleus. Leica DMI 6000B fluorescencemicroscope (Leica Microsystems, Wetzlar, Germany) was then used toobserve cells after being mounted.

Reverse Transcriptase Polymerase Chain Reaction

Qiagen RNAeasy kit (Qiagen, NV, Venlo, Netherlands) was used to isolatetotal RNA and Verzo cDNA kit (Thermo Fisher Scientific) was used forreverse transcription. Polymerase chain reaction (PCR) was carried outin 25 μL reaction mixtures. These reaction mixtures contained 1.0 μL ofcDNA, 1× Qiagen buffer, 0.2 mM of dNTP mixture, 0.2 μM of each primer,and 1.5 U of HotStar Taq (Qiagen, Valencia, Calif., USA). One cyclereaction at 95° C. for 15 minutes was followed by 35 amplificationcycles (94° C. for 1 minute, 66° C. for 1 minute, and 72° C. for 1minute) for MUC1. For β-actin PCR, the same conditions except for theannealing temperature (59° C.) were used. Primer sequences of MUC1 areforward 5′-TCTCAAGCAGCCAGCGCCTGCCTG-3′ (SEQ ID NO:2), and reverse5′-TCCCCAGGTGGCAGCTGAACC-3′ (SEQ ID NO:3). Primer sequences of β-actinare forward 5′-CCAAGGCCAACCGCGAGAAGAT-3′ (SEQ ID NO:4), and reverse5′-TTGCTCGAAGTCCAGGGCGA-3′ (SEQ ID NO:5).

Flow Cytometry

Approximately 1 million cells per well (A549 and MRC-5) were seeded in asix-well plate and incubated for 48 hours. Cells were then treated with100 m/mL of siGLO-FAM-loaded equivalent nanoparticles resuspended inOpti-MEM medium and incubated for 4 hours. Corresponding cells werepretreated with five times excess of free MUC1 aptamer, 60 minutes priorto being treated with the MUC1 aptamer-functionalized siGLO-FAM-loadedhybrid nanoparticles. Cells were washed with PBS. The cells were thendetached by trypsinization and centrifuged at 1,000 rpm for 5 minutes.The pellet was washed and resuspended in PBS. A 0.75 μm cell strainerwas used to filter the cells before being analyzed by flow cytometry(BDFACS caliber) to prevent cell aggregates from blocking the tube linesof the instrument. A total of 10,000 cells were measured in each sample.

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)Analysis

TUNEL assay was performed with TACS 2TdT-Fluor in Situ ApoptosisDetection Kit (Trevigen, Gaithersburg, Md.) according to manufacturer'sinstruction. Lung tissue section slides were deparaffinised in xylene,100%, 95%, 70% ethanol, followed by two changes of PBS. Samples werethen digested with 50 μl of Cytonin solution for 30 min and washed withwater and TdT Labelling Buffer. The slides were then covered with 50 μlof labelling reaction mix, which was then incubated for 60 min at 37° C.in a humidity chamber. Positive control was generated for comparison byincubating the slide with TACS-Nuclease (generating DNA breaks in everycell) at room temperature for 40 min, which was then followed by thelabelling step. Stop Buffer was then used to halt the labellingreaction, samples washed in PBS and covered with 50 μl ofStrep-Fluorescein Solution for 20 min. Slides were then washed with PBS,mounted and viewed under fluorescence microscope at 495 nm.

Cell Death Detection ELISA

Cell death detection ELISA was carried out according to manufacturer'sinstructions (Hoffman-La Roche Ltd., Basel, Switzerland). Briefly, A549cells were seeded at a density of 2×10⁴/well for 24 hours. Next day theywere treated with MUC1 aptamer-functionalized miR-29b-loaded hybridnanoparticles, MUC1 aptamer-functionalized negative control miRNA-loadedhybrid nanoparticles, and miR-29b in Lipofectamine 2000 TransfectionAgent (Ambion, Austin, Tex., USA) in Opti-MEM medium. Cells were washedin PBS and lysed in 200 μL/well of incubation buffer for 30 minutes atroom temperature. The lysates were centrifuged at 4,000×g for 10minutes. Supernatant was used right away in the experiment. Plasticwells of the ELISA kit were incubated with coating solution (containingantihistone antibodies) overnight at 4° C. Next day it was substitutedby incubation buffer for 30 minutes and washed three times with washingsolution. A total of 100 μL of homogenate was placed into the wells andincubated for 90 minutes at room temperature with mild shaking. It waswashed three times and incubated with conjugate solution containinganti-DNA peroxidase antibodies for 90 minutes. Wells were washed threetimes and exposed to ABTS substrate for 30 minutes until the colordeveloped. Absorption values were read using a BioTek Epoch MicroplateSpectrophotometer (BioTek Instruments Inc., Winooski, Vt., USA) withGen5 1.10 software.

Western-Blot Analysis

Tissue samples were collected from the lungs of treated mice. Thesesamples were snap-frozen in liquid nitrogen and kept at −80° C. untilready to be used. Tissue lysates were prepared in Pierce® RIPA buffer(Thermo Scientific, Rockford, Ill.) with addition of PIERCE™ ProteaseInhibitor Mini Tablets (Thermo Scientific, Rockford, Ill.). Proteinconcentration was determined with the COOMASSIE PLUS™ (Bradford) AssayKit (Thermo Scientific, Rockford, Ill.). 80 μg of total proteins wasseparated on NuPAGE 4-12% Bis-Tris gels (Life Technologies, Carlsbad,Calif.) with NuPAGE MES SDS running buffer (Life Technologies, Carlsbad,Calif.) and subsequently transferred onto nitrocellulose membrane of0.45 μm pore size (Life Technologies, Carlsbad, Calif.). Blocking wascarried out according to manufacturer's instructions (Invitrogen,Carlsbad, Calif.) for 1 hour at room temperature and probed with Rabbitantihuman DNMT3B monoclonal antibody (1:500) and Mouse antihumanβ-tubulin antibody (1:5000) overnight at 4° C. The membranes were washed5 min, thrice in wash buffer (Invitrogen, Carlsbad, Calif.). Membraneswere then incubated with secondary goat anti-mouse antibodies,conjugated with horseradish peroxidase (Molecular Probes, Eugene, Oreg.)at a dilution 1:1000. Immune complexes were detected Pierce ECL WesternBlotting Substrate (Thermo Scientific, Rockford, Ill.) in a dark room ontable top processor SRX-101A (Konica Minolta, Japan). Images werequantified in the Image J software.

Western Blot Analysis

One million A549 cells per well were seeded in culture dish andincubated for 48 hours. Cells were treated with MUC1aptamer-functionalized miR-29b-loaded hybrid nanoparticles (at anequivalent concentration of 100 nM of miRNA-29b), MUC1aptamer-functionalized negative control miRNA-loaded hybridnanoparticles as well as miR-29b in Lipofectamine 2000 reagent inOpti-MEM medium for 3 days. Cells were lysed in Pierce® RIPA buffer(Thermo Fisher Scientific) with addition of Pierce™ Protease InhibitorMini Tablets (Thermo Fisher Scientific) for 30 minutes on ice andcentrifuged for 10 minutes at 10,000× g at +4° C. The proteinconcentration was determined with the Coomassie Plus™ (Bradford) AssayKit (Thermo Fisher Scientific). A total of 120 μg of total proteins wasseparated on NuPAGE 4%-12% Bis-Tris gels (Thermo Fisher Scientific) withNuPAGE MES SDS running buffer (Thermo Fisher Scientific) andsubsequently transferred onto a nitrocellulose membrane with a pore sizeof 0.22 μm (Thermo Fisher Scientific). The membrane with proteins wasblocked according to manufacturer's instructions (Thermo FisherScientific) for 1 hour at room temperature and probed with primaryantibodies overnight at 4° C. DNMT3B (1:1,000, Thermo Fisher Scientific)and MCL1 (1:500; Thermo Fisher Scientific). β-Tubulin was used as ahousekeeping gene (1:2,000, Sigma-Aldrich). Next day the membranes werewashed three times for 5 minutes in wash buffer according tomanufacturer's instructions (Thermo Fisher Scientific) and incubatedwith secondary goat antimouse (Molecular Probes, Eugene, Oreg.) or goatantirabbit (1:1,000, Thermo Fisher Scientific) secondary antibodies,conjugated with horseradish peroxidase at a dilution of 1:1,000.

Immune complexes were detected with chemiluminescent substrate, PierceECL Western Blotting substrate (Thermo Fisher Scientific), in a darkroom on tabletop processor SRX-101A (Konica Minolta, Tokyo, Japan).

Cell Viability

MTT assay was used to determine the effect of MUC1aptamer-functionalized miRNA-29b-loaded hybrid nanoparticles on theproliferation of A549 cells. Cells (1×10⁴) per well were seeded in96-well plates and incubated at 37° C. in a humidified atmosphere with5% carbon dioxide for 24 hours. The cells were then treated withdifferent concentrations of miRNA-29b-loaded hybrid nanoparticles inOpti-MEM for 7 hours before being replaced with F12 K mediumsupplemented with 10% FBS and 1% antibiotics. This was then incubatedfor 72 hours. miRNA-29b transfected with NeoFX transfection agent wasused as control in this experiment. Approximately 10 μL of 12 mmol/L MTTreagent was then added to each well. This was then incubated at 37° C.for 4 hours. The medium was aspirated, and 50 μL of sterile dimethylsulfoxide were added to each well and mixed thoroughly with pipette. Thecells were then incubated at 37° C. for 10 minutes. The plate was readat 540 nm and 650 nm.

Animals

Female severe combined immunodeficient (SCID) beige mice, 8 weeks oldwere obtained from Taconic Co. (Hudson, N.Y.). These mice weighedapproximately 25 g.

Animal Treatment and Blood Sampling for Pharmacokinetic andBiodistribution Studies.

Female SCID beige mice were injected with 5×10⁵ A549-luciferase cellssuspended in sterile PBS to create metastatic models of NSCLC. XenogenIVIS bioluminescence imaging system was used to monitor tumor growth byadministering 100 μl of 30 mg/ml Xenolight Rediject D-Lucifereinintraperitoneally into the mice approximately ten minutes beforeimaging. Once tumors were established, tumor-bearing mice were dividedinto three groups of three animals each. The first group was treatedwith MAFMILHN containing an equivalent miRNA-29b dose of 1.5 mg/kg twiceweekly. The second group was treated with MUC1-aptamer functionalizedcontrol miRNA-loaded hybrid nanoparticles (NC-nano) containing anequivalent control miRNA dose of 1.5 mg/kg twice weekly byintraperitoneal injection. The third group was treated with PBS twiceweekly also, by peritoneal injection. Nanoparticles were dispersed insterile PBS. All mice were treated for a total of four weeks.

Tumor burden was monitored using Xenogen IVIS bioluminescence imagingsystem. For biodistribution and pharmacokinetic study, tumor bearingSCID beige mice were divided into two groups of three animals each. Thefirst group was treated with a single dose of MAFMILHN loaded with anequivalent dose of 1.5 mg/kg miRNA-29b. The second group was treatedwith a single dose of non-functionalized miRNA-29b loaded hybridnanoparticles loaded with an equivalent dose of 1.5 mg/kg. Blood sampleswere collected from each group per time point at 0, 30 and 60 min, and24, 26 and 48 h post-dose by retro-orbital puncture into EDTA tubes.

Animals were also sacrificed 48 h posttreatment to harvest the lungs,kidneys, heart and livers for analysis. Blood samples were mixed withequal amount of Lysis-loading buffer of Clarity OTX kit (Phenomenex, CA)and stored at −80° C. until ready to be used. Tissues were snap-frozenin the liquid nitrogen stored at −80° C. before analysis.

Pharmacokinetic (PK) Data Analysis

WinNonlin software version 6.0 (Pharsight, Mountain View, Calif.) wasused for the estimation of PK parameters using non-compartment analysisof the composite data.

Hyperspectral Microscopy

Cells and lung tissue section slides were prepared for hyperspectralmicroscopy. Hyperspectral images were captured at 60× magnificationunder enhanced darkfield illumination using the CytoViva hyperspectralimaging system (Auburn, Ala.). This hyperspectral imaging system couplesan enhanced darkfield illuminator with a CCD attached to a visiblenear-infrared (VNIR) diffraction grating spectrograph. The hyperspectralimages, also called data cubes, were collected using the “push broom”method facilitated by an automated motorized stage. The spectral data(400-1000 nm) was acquired one pixel row at a time. A version of ENVIimaging and analysis software proprietary to CytoViva was used tocompile the spectral and spatial data into a data cube, in which eachpixel contained spectral data. Spectral libraries corresponding to thenanoparticles and tissue were built from the hyperspectral images of thefunctionalized and non-functionalized nanoparticles as well as theexposed tissue. These libraries were filtered against a negative controlimage (tissue only) to ensure no false-positive mapping of thenanoparticles. Using the Spectral Angle Mapper (SAM) algorithm, thespectral libraries were compared to their respective images. The pixelsin the exposed sample whose spectra matched a spectrum in the libraryfor the functionalized nanoparticles were pseudo-colored red, confirmingthe presence of the nanoparticles.

Statistical Analysis

Results are presented as mean±SD, unless otherwise indicated.Statistically significant difference between two groups was determinedby two-tailed Student's t-test. A P-value of 0.05 was taken asstatistically significant.

Example 1: Nanoparticle Characterization

Particle size and zeta potential analysis by photon correlationspectroscopy, shown in Table 1, demonstrated that non-functionalizedhybrid nanoparticles were about 240 nm in size, while MUC1aptamer-functionalized hybrid nanoparticles were about 595 nm in size.Non-functionalized hybrid nanoparticles were negatively charged with azeta potential of −2.1, while the conjugation of MUC1 aptamer increasedthe zeta potential of these nanoparticles to +4.1.

TABLE 1 Particle size and zeta potential of non-functionalized andaptamer-functionalized hybrid nanoparticles (mean ± SD, n = 3)Nanoparticle Mean diameter Polydispersity Zeta potential sample (nm)index (mV) Non- 236.2 ± 35.1 0.242 ± 0.007 −2.1 ± 1.7 functionalizednanoparticles Aptamer- 595.9 ± 43.1 0.554 ± 0.386 +4.1 ± 1.0functionalized nanoparticles

The schematic of the MUC1 aptamer-functionalized hybrid nanoparticle isshown in FIG. 1A. Scanning electron micrograph in FIG. 1B reveals thatthe hybrid nanoparticles were spherical in shape with smooth surface.

miRNA encapsulation efficiency (EE) and loading capacity (LC) weremeasured using ion-pair HPLC analysis of the filtrates obtained bycentrifuging the nanoparticles formed to determine the amount ofunencapsulated miRNA-29b. EE and LC were calculated using the followingequation:

% EE=(A−B)×100/A, and % LC=(A−B)×100/C  (1)

where A=total amount of miRNA, B=free miRNA, and C=weight of hybridnanoparticles in grams.

EE was calculated to be 98.8%±0.4%, whereas LC was calculated to be8.6%±0.1%.

Conjugation of MUC1 aptamer to the surface of the nanoparticles wasverified using fluorescence microscopy. FIG. 1C demonstrates thesuccessful conjugation of FITC-MUC1 aptamer to the nanoparticles by thepresence of fluorescent green color in the micrograph. In contrast,non-functionalized hybrid nanoparticles in FIG. 1D showed the absence offluorescent green color, confirming the lack of FITC-MUC1 aptamer in thenanoparticles. Fourier transform-infrared (FT-IR) was also used to toconfirm the successful conjugation of MUC1 aptamer to the nanoparticles.FIG. 1E shows the FT-IR spectra generated from functionalized andnon-functionalized hybrid nanoparticles. A distinctive and conspicuousdifference between these sets of spectra bears testament to thesuccessful conjugation of MUC1 aptamer to the hybrid nanoparticles.

Example 2: In Vitro Release Study

The release of miRNA-29b from MUC1 aptamer-functionalized hybridnanoparticles was compared at pH values 5, 6.6, and 7.4. FIG. 2demonstrates a limitation in the release of miRNA-29b at pH values 6.6and 7.4 when compared with the release profile at pH 5.

Example 3: Expression of MUC1 Protein in NSCLC Cells

Reverse transcriptase PCR was used to confirm the expression of MUC1 intwo NSCLC cells, A549 and H460, to ensure that these cells actuallyexpress MUC1. FIG. 3A demonstrates the relatively higher expression ofMUC1 in both cancer cells when compared with normal lung fibroblast cellline MRC-5.

Example 4: Cellular Uptake of MUC1 Aptamer-Functionalized HybridNanoparticles

Internalization of nanoparticles was evaluated using both flow cytometryand fluorescence microscopy. Flow cytometry was used to comparenanoparticle uptake by A549 and MRC-5. siGLO-FAM (green) was used as amodel small double-stranded RNA labeled with FAM (a green dye). MUC1aptamer conjugated to siGLO-loaded hybrid nanoparticles used in thisexperiment was bereft of FITC so as to avoid double fluorescence. Asshown in FIG. 3B, internalization of MUC1 aptamer-functionalizedsiGLO-FAM-loaded nanoparticles in A549 cells was significantly higher(P≤0.001) in non-inhibited cells when compared with cells pretreatedwith MUC1 aptamer. Furthermore, uptake of nanoparticles by MRC-5 wasmuch lower than the uptake by A549 cells.

Fluorescence microscopy was used to study the interaction of FITC-MUC1aptamer-functionalized hybrid nanoparticles with the cell membrane ofA549 cells. FIG. 4A demonstrates the presence of FITC-MUC1aptamer-functionalized hybrid nanoparticles (green color) on the cellmembrane and inside the cytosol of A549 cells after 2 hours ofincubation. Cell membrane of cells was stained with WGA-Alexa Fluor 555(red color) to enable the identification of the boundaries of cells.Without wishing to be limited by any theory, colocalization of bothgreen and red colors on the membrane of the cells suggests a possibleinteraction between the FITC-MUC1 aptamer-functionalized hybridnanoparticles and the membrane of the cells possibly due to the presenceof MUC1 on the membrane.

Intracellular trafficking of internalized nanoparticles was alsomonitored using fluorescence microscopy. Late endosomes/lysosomes werelabeled with LysoTracker-Red to enable the monitoring of the fate ofinternalized nanoparticles. FIG. 4B demonstrates maximum level ofcolocalization of siGLO-FAM and LysoTracker-Red after 2 hours ofincubation. However, after 4 hours, the siGLO-FAM was observed to beleaving the late endosomes and moving into the cytoplasm as indicated bythe numerous green colored dots surrounding the nucleus.

Cellular uptake was further monitored with hyperspectral microscopy(Cytoviva Inc. Auburn, Ala.). FIGS. 7A-7H show micrographs captured byhyperspectral microscopy. FIGS. 7A-7B demonstrate the sphericalmorphology of both MAFMILHN and non-functionalized miRNA-29b-loadedhybrid nanoparticles. This is consistent with the image of themorphology obtained by SEM in FIG. 61 . FIGS. 7C-7D show the untreatedA549 and MRCS cells respectively, showing lack of presence ofnanoparticles in the cells. However, FIG. 7E demonstrates the presenceof numerous MAFMILHN in A549 cells following 2 h-treatment. This is incontrast to A549 cells in FIG. 7F treated with non-functionalized hybridnanoparticles, showing limited amount of internalized nanoparticles.This demonstrates the influence of MUC1-aptamer in the uptake of thesenanoparticles. FIGS. 7G-7H demonstrate that both MAFMILHN andnon-functionalized hybrid nanoparticles were not taken up significantlyby MRCS due to the limited expression of MUC1 in this cell.

Example 5. Tissue Distribution and Pharmacokinetic Study

Following the harvest of essential organs from euthanized lung-tumorbearing SCID beige mice, the deposition of miRNA-29b in the heart,tumor-bearing lungs, kidney and liver was quantified. FIGS. 8A-8Bdemonstrate the importance of MUC1-aptamer in ensuring that miRNA-29bloaded in MAFMILHN was preferentially delivered to tumor bearing lungswhile limiting accumulation in healthy organs such as kidney, liver andheart. In the absence of MUC1-aptamer, as demonstrated in FIG. 8B,non-selective delivery of miRNA-29b was observed by non-functionalizedhybrid nanoparticles with more miRNA-29b detected in the liver andkidney than in the lungs. This demonstrates the importance ofMUC1-aptamer in targeted delivery of miRNA-29b to lung tumor withminimal delivery to other organs. To visualize the presence ofnanoparticles in the lungs, tissue sections of lungs obtained from thetwo experimental groups were imaged using hyperspectral microscopy. FIG.8C shows high deposition of MAFMILHN in lung tumor of mice treated withthese nanoparticles. In contrast, lung tumor of mice treated withnon-functionalized miRNA-29b loaded hybrid nanoparticles show limitedamount of nanoparticles (FIG. 8D), further demonstrating the selectiveand specific delivery of miRNA-29b using MUC1-aptamer functionalizedhybrid nanoparticles.

FIG. 9 demonstrates the plasma concentration versus time curves whenequivalent of 1.5 mg/kg of miRNA-29b in MAFMILHN. The maximumconcentration (C_(max)) of miRNA-29b delivered MAFMILHN was 15289.5ng/ml, slightly lesser than 18289.5 ng/ml observed for thenon-functionalized miRNA-29b-loaded hybrid nanoparticles. These maximumconcentrations were observed at 60 mins (T_(max)) for bothnanoparticles.

Key pharmacokinetic parameters presented in Table 2 further demonstratethe similarities in the pharmacokinetics of both MAFMILHN andnon-functionalized miRNA-29b-loaded hybrid nanoparticles. Both MAFMILHNand non-functionalized miRNA-29b-loaded hybrid nanoparticles produced ahalf-life of 13.3 and 14.6 h respectively. Further, both formulationsproduced similar men residence time (MRT) and apparent clearance.

TABLE 2 Key pharmacokinetic parameters of miRNA-29b loaded in MAFMILHNand non-functionalized hybrid nanoparticles in orthotopic SCID models ofNSCLC. Non-functionalized Parameters MAFMILHN hybrid nanoparticlesC_(max) ng/ml 15289.5 18289.5 T_(max) (h) 1.0 1.0 AUC_(last) (h · ng/ml)443991.8 540341.8 CL/F (ml/min/kg) 5.04 × 10⁻⁸ 4.04 × 10⁻⁸ λ_(z) (1/h)0.05208 0.04763 T_(1/2) (h) 13.3 14.6 MRT (h) 16.4 16.7 Vz/F (l/kg)  5.8× 10⁻⁵  5.1 × 10⁻⁵ C_(max) = Peak plasma concentration; T_(max) = Timeto peak plasma concentration; AUC_(last) = area under the plasmaconcentration-time curve from time zero to time of last measurableconcentration; CL/F = Apparent clearance; λ_(z) = Elimination rateconstant; T_(1/2) = Plasma terminal half-life; MRT = Mean residencetime; Vz/F = Apparent volume of distribution.

Example 6: MUC1 Aptamer-Functionalized miRNA-29b-Loaded HybridNanoparticles Downregulate DNMT3B and MCL1 Proteins and Induce Apoptosisin A549 Cells

FIG. 5A demonstrates the knockdown efficiency of MUC1aptamer-functionalized miRNA-29b-loaded nanoparticles against DNMT3B.These nanoparticles produced superior downregulation of DNMT3B whencompared with Lipofectamine 2000 transfected miRNA-29b and the negativecontrol miRNA-loaded nanoparticles. FIG. 5B further demonstrates thesuperior transfection efficiency of MUC1 aptamer-functionalizedmiRNA-29b-loaded nanoparticles in comparison to lipofectamine byeffectively downregulating MCL1. In contrast, both lipofectamine andMUC1 aptamer-functionalized negative control miRNA-loaded hybridnanoparticles did not achieve the same level of downregulation.

Superior induction of apoptosis was observed in A549 cells treated withMUC1 aptamer-functionalized miRNA-29b-loaded hybrid nanoparticles whencompared with non-treated cells, MUC1 aptamer-functionalized negativecontrol miRNA-loaded hybrid nanoparticles-treated cells andlipofectamine-transfected miRNA-29b (FIG. 5C).

Antiproliferative effect of MUC1 aptamer-functionalized miRNA-29b-loadednanoparticles was evaluated using MTT assay and compared with that ofNeoFX-transfected miRNA-29b and negative control miRNA-loadednanoparticles in A549 cells. As demonstrated in FIG. 5D, MUC1aptamer-functionalized miRNA-29b-loaded nanoparticles were significantlymore cytotoxic to A549 cells than NeoFX-transfected miRNA-29b andnegative control miRNA-loaded miRNA nanoparticles.

The ability of MAFMILHN to induce apoptosis in lung tumor was evaluatedusing both TUNEL assay and cell death-detection ELISA. The lungs oftumor-bearing mice treated with MAFMILHN demonstrated high level ofapoptosis as measured by TUNEL in FIG. 10A. Conversely, mice treatedwith NC-nano and PBS demonstrated very low level of apoptosis. Resultsgenerated form TUNEL assay was validated with cell death-ELISA datapresented in FIG. 10B. Similar to the TUNEL results, cell death ELISAshowed that apoptosis in the lungs of mice treated with MAFMILHN wassignificantly higher than in the lungs of mice treated with NC-nano andPBS.

Tumor suppression capability of MAFMILHN was evaluated using IVISbioluminescence imaging system to monitor tumor burden in tumor-bearingSCID mice over a four-week period. FIGS. 11A-11B demonstrate the abilityof MAFMILHN to suppress tumor growth when compared to differentcontrols. In FIG. 11A, the intensity of bioluminescence was quantifiedand plotted against time. While the tumor in mice treated with NC-nanoand PBS continued to grow over the study period, tumor in mice treatedwith MAFMILHN decreased in intensity over the same period.Representative bioluminescence images are shown in FIG. 11B. Theseimages are constituent with the graphical presentation in FIG. 11A.

Example 7

As demonstrated herein, the ability of MUC1-aptamer functionalizedmiRNA-29b-loaded hybrid nanoparticles (MAFMILHN) to selectively delivermiRNA-29b to lung tumor while limiting accumulation in healthy tissueswas demonstrated. The described pharmacokinetic (PK) studies helpeddetermine some critical PK parameters for MAFMILHN.

MAFMILHN nanoparticles were prepared, and conjugation of MUC1-aptamer tothe hybrid nanoparticles increased the particle size from 236 nm to 595nm. Without wishing to be limited by any theory, the change in size canbe at least partially explained by the presence of the aptamer on thesurface of the nanoparticles, along with the extra steps involved in theconjugated process, especially the use of lyophilization, which couldlead to agglomeration of particles. This is further reflected in thepolydispersity index (PDI) increase from 0.243 to 0.554 following theaptamer conjugation procedure.

Intracellular delivery of the nanoparticles in both A549 cells and lungtumor tissues were probed using hyperspectral microscopy. Hyperspectralmicroscopy, which was developed to address current analytical challengesfor nanoscale materials, combines hyperspectral imaging (HSI) withadvanced optics, typically focusing on specialized dark-fieldreflectance systems. Hyperspectral microscopy data in FIGS. 7A-7Hconfirms the selective delivery of MAFMILHN to mucin expressing cells incomparison to cell with limited mucin-1 expression. A549 cells showedvery high level of expression of mucin-1 as compared to MRC-5, whichshowed very limited level of mucin-1. Hyperspectral microscopy showedthat MAFMILHN were preferentially delivered to A549 cells when comparedto MRC-5. This correlates with the comparative level of mucin-1 in bothcells. Further, hybrid nanoparticles that were not conjugated withMUC1-aptamer were not efficiently delivered to either A549 cells orMRC-5.

The effect of MUC1-aptamer on the selective delivery of MAFMILHN totumor tissues in mouse models and compared to non-functionalized hybridnanoparticles. MAFMILHN were found to favorably deliver miRNA-29b totumor-bearing lung tissues in contrast to heart liver and kidney in FIG.8A. However, the non-functionalized hybrid nanoparticles were not ableto achieve this in FIG. 8B. Further, hyperspectral microscopy images inFIGS. 8C-8D were able to confirm that MAFMILHN were indeed accumulatedin tumor bearing lung tissue, while such could not be said fornon-functionalized hybrid nanoparticles. The liver and kidneys also hadminor amounts of miRNA-29b delivered to them by MAFMILHN. The liveramongst other organs such as spleen belongs to the reticuloendothelialendothelial system organs. In certain non-limiting embodiments, theliver, being a highly perfused organ, enables rapid distribution ofnanoparticles to this organ, whether the nanoparticle is targeted ornot. In addition, the microvessels of liver have relatively largefenestrations, which allow entry of particles as high as approximately200 nm. The kidney also demonstrated a limited amount of miRNA-29b fromMAFMILHN; in certain non-limiting embodiments, this organ is highlyperfused and the main organ for elimination. Although limited amount ofmiRNA-29b was delivered to tumor-bearing lungs by non-functionalizedhybrid nanoparticles regardless of these nanoparticles not beingfunctionalized by MUC1-aptamer.

The pharmacokinetic parameters of both MAFMILHN and that of thenon-functionalized hybrid nanoparticles were very similar, suggestingthat, although the conjugation of MUC1-aptamer to the nanoparticles inMAFMILHN enhanced the discriminatory delivery of miRNA-29b to lung tumortissues, it did not influence the peak plasma concentration (C_(max)),time to peak plasma concentration (T_(max)), area under the plasmaconcentration-time curve from time zero to time of last measurableconcentration (AUC_(last)), apparent clearance (CL/F), elimination rateconstant (λ_(z)), plasma terminal half-life (T_(1/2)), mean residencetime (MRT) and apparent volume of distribution to any significant extentwhen compared to the non-functionalized hybrid nanoparticles. In certainnon-limiting embodiments, the presence of the nanoparticles in bothformulations protect the loaded miRNA-29b from marauding endonucleaseenzymes and phagocytes, hence helping to enhance the circulation time ofmiRNA-29b in the blood.

To evaluate the ability of MAFMILHN to downregulate target oncoproteinDNMT3B in both in vitro and in vivo models, its expression was monitoredusing western blot. As demonstrated in FIG. 5A, DNMT3B was downregulatedin A549 cells following treatment with MAFMILHN in a superior versionwhen compared to lipofectamine-transfected miRNA-29b as well asMUC1-aptamer functionalized control miRNA-29b-loaded hybridnanoparticles. This result was consisted in SCID mice (in vivo), asdemonstrated by the result in FIG. 5B in which MAFMILHN was able tocomplete downregulate the expression of DMNT3B. DNMT3B is a member ofthe DNA methyltrasferase family that accounts for the inactivation oftumor-suppressor genes in many cancer cells. However, miRNA-29b is knownto exert its tumor-suppressive role by directly targeting DNMT3B incancer cells. Without wishing to be limited by any theory,downregulation of DNMT3B can lead to the induction of apoptosis in tumortissues, hence leading to the inhibition of tumor growth in SCID mousemodels. FIGS. 10A-10B and 11A-11B indicate that apoptosis was observedin treated mice, which consequently led to the inhibition of tumorgrowth in the animals.

Taken together, the present results indicate that MUC1-aptamerfunctionalized hybrid nanoparticles can be used as a platform targetednanoparticle delivery system for efficient delivery of miRNAs,especially miRNA-29b to non-small cell lung cancer for thedownregulation of target oncogene.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. One skilled in the art will readily appreciate that thepresent invention is well adapted to carry out the objects and obtainthe ends and advantages mentioned, as well as those inherent therein.While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope used in the practice of thepresent invention. The appended claims are intended to be construed toinclude all such embodiments and equivalent variations.

What is claimed:
 1. A protein-containing nanoparticle, wherein the coreof the nanoparticle comprises at least one protein selected from thegroup consisting of a plasma protein, an IgG, a cytokine, animmunomodulator, an antigen, a hormone, and an enzyme, wherein the atleast one protein is in a neutral state in the nanoparticle, wherein thenanoparticle is surrounded by a layer comprising a stealth polymer,wherein the at least one protein is conjugated with at least one cellsurface receptor ligand, wherein at least a fraction of the ligand isdisplayed on the outer surface of the surrounding layer of thenanoparticle.
 2. The nanoparticle of claim 1, wherein the stealthpolymer is at least one selected from the group consisting of an alkylpolyethylene glycol, an alkylphenol oxide, a copolymer of polyethyleneglycol and polypropylene oxide, a polyethylene glycol, a polypropyleneglycol, a polyvinylpyrrolidone (PVP), a polyvinyl alcohol, or anycombinations thereof.
 3. The nanoparticle of claim 1, which has adiameter ranging from about 10 nm to about 1,000 nm.
 4. The nanoparticleof claim 1, wherein the plasma protein is at least one selected from thegroup consisting of albumin, fibrinogen, and globulin, and wherein thecytokine comprises at least one selected from the group consisting ofinterleukin, erythropoietin, interferon, and filgrastim.
 5. Acomposition comprising at least one protein nanoparticle of claim 1,which is prepared by a method comprising: adjusting the pH of a firstsolution comprising a protein to about the isoelectric point of theprotein, thereby forming a first protein nanoparticle, which comprisesat least a fraction of the protein; wherein, if the protein in the firstprotein nanoparticle is not conjugated to at least one cell surfacereceptor ligand, the protein in the first protein nanoparticle isfurther conjugated with the at least one cell surface receptor; and,contacting the first protein nanoparticle with a second solutioncomprising a stealth polymer, wherein the concentration of the stealthpolymer in the second solution ranges from about 0.1% to about 20,000%of the CMC of the stealth polymer, thereby forming at least one proteinnanoparticle.
 6. The composition of claim 5, wherein the proteincomprises at least one selected from the group consisting of a plasmaprotein, an IgG, a cytokine, an immunomodulator, an antigen, a hormone,and an enzyme.
 7. The composition of claim 5, wherein the first solutionfurther comprises at least one therapeutic agent, and wherein the firstprotein nanoparticle comprises at least a fraction of the at least onetherapeutic agent.
 8. The composition of claim 7, wherein the at leasttherapeutic agent is selected from the group consisting of an organiccompound, inorganic compound, pharmacological drug, antibody,radiopharmaceutical, protein, peptide, polysaccharide, nucleic acid,siRNA, miRNA, RNAi, short hairpin RNA, antisense nucleic acid, ribozymeand dominant negative mutant.
 9. The composition of claim 8, wherein theantibody comprises a monoclonal antibody selected from the groupconsisting of bevacizumab, anatumomab, benralizumab, enokizumab,mitumomab, oxelumab, and palivizumab.
 10. The composition of claim 7,wherein at least a fraction of the at least one cell surface receptorligand is displayed on the outer surface of the stealth polymer coatingof the nanoparticle, and optionally wherein the at least one cellsurface receptor ligand binds to at least one selected from the groupconsisting of neurotensin receptor-1, human epidermal growth factorreceptor-2 (HER-2), folate receptor, insulin-like growth receptor (IGF),and epidermal growth factor receptor (EGFR).
 11. The composition ofclaim 7, wherein the stealth polymer comprises at least one selectedfrom the group consisting of an alkyl polyethylene glycol, analkylphenol oxide, a copolymer of polyethylene glycol and polypropyleneoxide, a polyethylene glycol, a polypropylene glycol, apolyvinylpyrrolidone (PVP), a polyvinyl alcohol, or any combinationsthereof.
 12. The composition of claim 11, wherein the alkyl polyethyleneoxide comprises at least one selected from the group consisting of adiethylene glycol hexadecyl ether, polyethylene glycol oleyl ether,diethylene glycol octadecyl ether, polyoxyethylene stearyl ether,polyethylene glycol hexadecyl (cetyl) ether, polyethylene glycol dodecyl(lauryl) ether, decaethylene glycol oleyl ether, polyethylene glycoloctadecyl ether, and polyethylene glycol octadecyl ether.
 13. A methodof preparing at least one stealth polymer-coated protein nanoparticle,the method comprising: adjusting the pH of a first solution comprising aprotein to about the isoelectric point of the protein, thereby forming afirst protein nanoparticle, which comprises at least a fraction of theprotein; wherein, if the protein in the first protein nanoparticle isnot conjugated to at least one cell surface receptor ligand, the proteinin the first protein nanoparticle is further conjugated with the atleast one cell surface receptor; and, contacting the first proteinnanoparticle with a second solution comprising a stealth polymer,wherein the concentration of the stealth polymer in the second solutionranges from about 0.1% to about 20,000% of the CMC of the stealthpolymer, thereby forming the at least one stealth polymer-coated proteinnanoparticle.
 14. A method of treating, ameliorating or preventing adisease or disorder in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutically effective amount of ananoparticle of claim 1, further wherein the composition is administeredto the subject by an intrapulmonary, intrabronchial, inhalational,intranasal, intratracheal, intravenous, intramuscular, subcutaneous,topical, transdermal, oral, buccal, rectal, pleural, peritoneal,vaginal, epidural, otic, intraocular, or intrathecal route, optionallywherein the composition further comprises at least one therapeutic agentwithin the protein nanoparticle,
 15. The method of claim 14, wherein theat least therapeutic agent is selected from the group consisting of anorganic compound, inorganic compound, pharmacological drug, antibody,radiopharmaceutical, protein, peptide, polysaccharide, nucleic acid,siRNA, RNAi, short hairpin RNA, antisense nucleic acid, ribozyme anddominant negative mutant.
 16. The method of claim 14, wherein the atleast therapeutic agent comprises a siRNA or miRNA, and wherein theprotein comprises IgG.
 17. The method of claim 14, wherein the at leastone cell surface receptor ligand binds to at least one selected from thegroup consisting of neurotensin receptor-1, human epidermal growthfactor receptor-2 (HER-2), folate receptor, insulin-like growth receptor(IGF), and epidermal growth factor receptor (EGFR).
 18. The method ofclaim 14, wherein the disease or disorder is selected from the groupconsisting of colon cancer, rectum cancer, lung cancer, glioblastoma,renal cell cancer, non-small cell lung cancer, small cell lung cancer,asthma, respiratory syncytial virus (RSV) infection, and anycombinations thereof.
 19. A kit comprising a composition comprising ananoparticle of claim, the kit further comprising an applicator; and aninstructional material for the use of the kit, wherein the instructionmaterial comprises instructions for treating, ameliorating or preventinga disease or disorder in a subject in need thereof.
 20. A kit comprisinga stealth polymer, a protein conjugated with a cell surface receptorligand, an applicator, and an instructional material for the use of thekit, and wherein the instruction material comprises instructions forpreparing a stealth polymer-coated protein nanoparticle wherein at leasta fraction of the ligand is displayed on the outer surface of thestealth polymer coating of the nanoparticle.